Compound for treating clostridium difficile

ABSTRACT

The invention relates to compounds, compositions and polymers comprising a first component adapted to promote germination of Clostridium difficile (C.diff) and a second component which acts as an antimicrobial agent. Said compounds, compositions and polymers are useful for destroying C.diff where conventional antimicrobial agents are unsuccessful. The compositions can be formulated as coating or materials which actively destroy C.diff which come into contact with it. The germination promotion is induced by bile salts. The invention also relates to the use of such materials as a treatment for C.diff associated diseases and toxic megacolon.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage Application filed under 35U.S.C. § 371 and claims the benefit of International Application No.PCT/GB2015/053614, filed Nov. 26, 2015, which claims priority under 35U.S.C. § 119 to Great Britain Application No. 1421071.0, filed Nov. 27,2014, the disclosures of which are incorporated herein by reference.

FIELD OF INVENTION

The invention relates to a compound suitable for the treatment ofClostridium Difficile (C.diff.) and in particular, the use of conjugatedbile salts and polymers comprising bile salts.

BACKGROUND TO THE INVENTION

The spore forming bacterium Clostridium Difficile (C. diff.) is known tocause a variety of problems in the human body and particularly in thehuman digestive system. The bacterium in its spore form is extremelyresilient to antimicrobial agents as well as other forms of chemical andphysical treatments. These C. diff. spores are capable of surviving theharsh acidic conditions within the stomach and can end up in theintestines of the body.

Once in the intestine, typically the large intestine, C. diff. sporesare able to germinate into the more active bacterial species whichsecretes harmful toxins which cause illness such as pseudo-membranouscolitis or even toxic megacolon.

In order to destroy such spores outside the body, for example where thespores are present on surfaces, strong chemical cleaning agents such asbleach are required. This is undesirable as such cleaning agents (oftenused in high concentrations) are more difficult to handle compared tostandard antibacterial treatments and also can not be used on the skin.

However, the most significant problem with C. diff. infection appears tobe where a patient has contracted another, accompanying bacterialinfection and is treated with antibiotics, especially broad-spectrumantibiotics. The antibiotics destroy not only the target bacteria forwhich the antibiotics were prescribed, but also significantly reduce theamount of bacteria present in the gut. As C. diff. in its spore formtypically resist attack by antibiotics, this leads to a situation whereC. diff. spores can enter the gut, germinate into the more activebacterial species, and can thrive as there is little in the way ofcompetition from native gut bacteria.

These problems mean that C. diff. can be a significant problem inhospitals and healthcare facilities where many patients are undergoingor have recently completed a course of antibiotics and where healthcareprofessions, although diligent in their use of antimicrobial cleaningproducts to destroy conventional microbes present on their person inbetween patient interactions, may still transmit C. diff. spores to manyindividuals.

Current treatments for C. diff. infections include administration ofantibiotics but patients often experience relapses, possibly because theantibiotics are only capable of treating the germinated C. diff.bacteria not any residual ungerminated spores. Alternative treatmentsinclude performing a stool transplant in order to re-establish thebacterial environment of the gut, thereby reducing the amount of C.diff. bacteria due to increased competition for nutrients with thetransplanted bacteria. However, this procedure is not an attractiveoption for many patients.

Extensive research has been performed to examine what causes germinationof C. diff. spores. Wilson K (J. Clin Microbial 1983 1017-1019) statesthat the presence of certain bile salts appears to trigger germinationof C. diff. spores in the large intestine. Other sources such as Sorg JA and Sonenshein A L (J. Bacteriol 2008, 2505-2512) states that bothbile salts and certain amino acids play an important role in bringingabout germination of C. diff. spores. There has also been some researchinto compounds which act as inhibitors to the germination of C. diff.spores. Sorg J A and Sonenshein A L (J. Bacteriol 2010, 4983-4990) forinstance, discloses various compounds found to reduce the rate ofgermination of C. diff. spores.

Accordingly, what is required is a method of treating C. diff. spores invivo as well as methods for preventing the spread of C. diff. spores exvivo, particularly in healthcare environments.

The invention is intended to overcome or at least ameliorate some of theproblems outlined above.

SUMMARY OF THE INVENTION

There is provided in a first aspect of the invention, a compoundaccording to general Formula 1 or a pharmaceutically acceptable saltsthereof:X—Z-M  Formula 1wherein; X is a bile salt, Z is a linker and M is an antimicrobialagent.

The inventors have found that by coupling a bile salt compound to anantimicrobial agent, it is possible to stimulate germination of C. diff.spores to form the active bacterial species which, being now morevulnerable to attack, are subsequently destroyed by the accompanyingantimicrobial agent.

As bile salts are native to the body, produced by the liver and storedin the gallbladder, many of these compounds exhibit goodbiocompatibility and are relatively low in toxicity. The term “bilesalt” as used herein is intended to refer to the compounds having ageneral structure or skeletal structure similar to that of the naturallyoccurring bile salts produced by the human body. This includesstereoisomers of naturally occurring bile salts and also includesnaturally occurring bile salts comprising modified functional groups.

Typically, X is a compound according to formula 2:

wherein, R¹ is selected from: hydroxy, alkoxy, acyloxy, aryloxy,acrylate or methacrylate; R² and R³ are each independently selectedfrom: hydrogen, hydroxy, alkoxy, acyloxy, aryloxy, acrylate ormethacrylate groups.

Even more typically, X is a compound according to general formula 3:

wherein R¹, R² and R³ are as defined above and linker Z is attached tothe carbon at the terminal C(O) moiety.

The inventors have found that bile salts having the stereochemistryshown in formula 3 are particularly effective at promoting germinationof C. diff. spores. Without being bound by theory, it is believe thatthis orientation provides the most compatible structure with receptors,enzymes or structures present on the C. diff. spores which promotesgermination.

For the avoidance of doubt, the invention is intended to encompass thosecompounds described herein as well as prodrugs and/or the biologicallyactive species resulting from metabolisation of said compounds. The term“prodrug” is intended to refer to compounds which, on administration tothe body, are converted into a compound as described herein.

It is often the case that R¹ is selected from: hydroxy, alkoxy andaryloxy groups and even more typically R¹ is a hydroxy or alkoxy group.Typically, R¹ is a hydroxy group.

In a typical embodiment, R² and R³ are each independently selected from:hydrogen, hydroxy, alkoxy or aryloxy groups. R² and R³ may eachindependently be selected from: hydrogen, hydroxy and alkoxy groups andit is typically the case that R² and R³ are each independently selectedfrom hydrogen and hydroxy groups.

The linker “Z” is typically a covalent linker. The term “covalentlinker” is intended to mean a compound which is covalently bonded at afirst position to the X group and which is also covalently bonded at asecond position to the M group. Typically the first and second positionsare located at points on the linker which are spaced apart from oneanother and it is often the case that the first and second positions arelocated at the two ends of the linker. Usually the linker is of agenerally linear configuration having two ends, the two ends being thetwo extreme most ends of the linker.

In the situation where the first linkage Z is —NR—, R is typicallyselected from hydrogen, alkyl, aryl, acyl, alkenyl or alkynyl.

The linker Z may also be selected from: alkyl, aryl, acyl, alkenyl,alkynyl, ethers, polyether, esters, polyesters, amides, polyamides,amines or combinations thereof. More typically, the linker Z is selectedfrom alkyl, alkenyl, ethers, polyethers or combinations thereof. Mosttypically the linker Z is polyethylene glycol and as such the compoundof the invention may have a structure as shown in formula 4a:

wherein n is an integer in the range of 1 and 10, and wherein X and Mare as defined above. Typically, n is an integer in the range of 1 and 5and most typically n is 1, 2 or 3.

Alternatively, the linker Z may comprise a first linkage A and a secondlinkage L. The order of the “A” and “L” linkages may be interchangeablei.e. the compound of the invention may be represented as “X-A-L-M” or“X-L-A-M”. Typically however, it is the case that the compound of theinvention is in the form “X-A-L-M”.

In the situation where the first linkage A is —NR—, R is typicallyselected from hydrogen, alkyl, aryl, acyl, alkenyl or alkynyl. It istypically the case that A is —NR—.

The term ‘aryl’ is intended to refer to an aromatic ring structure. Thismay include one or more fused rings and the ring or rings may eachindependently be 5-, 6-, 7-, 8- or 9-membered rings. Typically, the arylgroups will be a single aromatic ring and even more typically, the ringmay be a 5-, or 6-membered ring.

The term ‘acyl’ is intended to linear or cyclic carbon chains as well asbranched carbon chains having at least one carbonyl group.

The term ‘alkenyl’ is intended to refer to linear or cyclic carbonchains as well as branched carbon chains having at least one unsaturatedcarbon-carbon double bond.

The term ‘alkynyl’ is intended to refer to linear or cyclic carbonchains as well as branched carbon chains having at least one unsaturatedcarbon-carbon triple bond.

The term ‘alkyl’ is intended to encompass aliphatic, linear and cyclicsaturated carbon chains as well as branched saturated carbon chains.

The terms ‘alkyl’, ‘aryl’, ‘alkenyl’, ‘acyl’ and ‘alkynyl’ are intendedto encompass groups which may be substituted with one or moreheteroatoms and is also intended to encompass halogenated analoguesthereof.

Typically each the ‘alkyl’, ‘aryl’, ‘alkenyl’, ‘acyl’ and ‘alkynyl’ arein the range of C₁ to C₁₀, more typically between C₁ to C₈ and even moretypically C₁ to C₅ in length.

It is usually the case that R is selected from hydrogen or alkyl.Typically, R is a C₁-C₁₀ alkyl group which may be a linear alkyl group.

The second linker L is often selected from: alkyl, aryl, acyl, alkenyl,alkynyl, ethers, polyether, esters, polyesters, amides, polyamides,amines or combinations thereof. More typically, the linker L is selectedfrom alkyl, alkenyl, ethers, polyethers or combinations thereof. Mosttypically, the linker L is polyethylene glycol and as such the compoundof the invention may have a structure as shown in formula 4b or 4c:

wherein n is an integer in the range of 1 and 10, and wherein X, A and Mare as defined above. Typically, n is an integer in the range of 1 and 5and most typically n is 1, 2 or 3.

M is typically an antibacterial agent. The antibacterial agent may be abroad-spectrum antibacterial agent or may be a specific antibacterialagent. Often, M may be an antibiotic. The antibiotics may be suitablefor treating gram-negative bacteria and/or gram-positive bacteria.

In the invention, it is typically the case that M is a quaternaryammonium salt and M may be a compound according to general formula 5:

wherein each of R⁴, R⁵ and R⁶ are independently selected from: alkyl,aryl, alkenyl, alkynyl, alkylamides or alkylamines. Each of R⁴, R⁵ andR⁶ may be independently selected from: alkyl, aryl, acyl or alkenyl.

Usually, at least two of R⁴, R⁵ and R⁶ are covalently bonded togetherand it is often the case that two of R⁴, R⁵ and R⁶ are covalently bondedtogether. The counter ion accompanying the quaternary ammonium salt isnot particularly limited to a specific group. Typically, the counter ionwill be an inorganic anion and it is usually the case that the counterion is a chloride ion.

The inventors have found that quaternary ammonium salts function well asantibacterial agents. Without being bound by theory, it is believed thatunlike certain antimicrobial agents such as silver ions, the quaternaryammonium salts are not used up in the process of destroying the targetpathogen. It is though that the quaternary ammonium salt catalyses thedestruction of pathogens and is not used up, or at least is used up at amuch slower rate than other antimicrobial agents, in the process.

In a further embodiment of the invention, there is provided a compoundaccording to any one of the following formulae 6a-6n:

wherein n and m are each independently an integer between 1 and 10, andR⁷ is a bile salt as described above. The skilled person wouldunderstand that formulae 6a-n are intended to cover compounds whereinthe bile salt group R⁷ refers to the bile salt minus the—CCH₃(CH₂)₂CONH—.

In a second aspect of the invention, there is provided a compositioncomprising a compound according to the first aspect of the inventionfurther comprising one or more additives.

Typically the additives are selected from: amino acids, conjugated aminoacid, bile salts and/or antimicrobial agents.

The amino acids suitable for inclusion in the composition of theinvention may be any of the naturally occurring amino acids and it istypically the case that the amino acids and conjugated amino acids areselected from: glycine, glutamine, leucine, isoleucine, alanine andtaurine. It may be the case that the conjugated amino acid is taurine.

Often, the antimicrobial agents used as additives with the compositionof the invention are antibiotics. These antibiotics may bebroad-spectrum antibiotics or bacteria specific antibiotics. Usually,the antibiotics will be effective at treating C. diff. bacteria. Theantibiotics may be suitable for treating gram-negative and/orgram-positive bacteria.

In the composition of the invention, it is typically the case that theantimicrobial agent additive is a quaternary ammonium salt according togeneral Formula 7:

wherein each of R′, R⁴, R⁵ and R⁶ are independently selected from:alkyl, aryl, alkenyl, alkynyl. Each of R′, R⁴, R⁵ and R⁶ may beindependently selected from: alkyl, aryl, alkenyl.

Usually, at least two of R′, R⁴, R⁵ and R⁶ are covalently bondedtogether and it is often the case that two of R′, R⁴, R⁵ and R⁶ arecovalently bonded together. The counter ion accompanying the quaternaryammonium salt is not particularly limited to a specific group.Typically, the counter ion will be an inorganic anion and it is usuallythe case that the counter ion is a chloride ion.

In an alternative embodiment of the invention, there is provided acomposition comprising either a bile salt and an antimicrobial agent; ora compound, polymer or copolymer according to the above aspects of theinvention or combinations thereof. Where the composition comprises acompound, polymer or copolymer or combinations thereof according to theabove aspects of the invention, the composition may additionallycomprise a bile salt and/or an antimicrobial agent.

As mentioned above, the combination of bile salts and an antimicrobialagent provides both C. diff. spore germinating properties as well asantimicrobial properties that can destroy subsequently germinated C.diff. bacteria. Therefore, by including one or more bile salts, asdefined above, in a composition containing an antimicrobial agent,environments containing C. cliff. spores can be made safe by exposingsaid environments to the composition thereby forcing germination of theC. diff. spores which, in the presence of an antimicrobial agent, aresubsequently destroyed. This is particularly advantageous ex vivo inhealthcare environments.

Typically, the composition is a cleaning agent for cleaning surfaces andenvironments containing C. diff. spores. It is more difficult to treatC. diff. spores present within an infected patient as many antimicrobialagents suitable for use ex vivo are not suitable for in vivo use andalso because the bile salts and antimicrobial agents can not act intandem with one another due to the difficulties in getting bothcompounds into the gut together at the same time. This problem can besolved by using the compound according to the first aspect of theinvention which covalently links the bile salt and the antimicrobialcompounds together.

The cleaning agent may additionally comprise a compound, polymer,copolymer or combinations thereof according to the above aspects of theinvention as well as other more conventional additives. For example, thecleaning agent may further comprise one or more solvents. Typicallythese will be hydrophobic or hydrophilic solvents. The cleaningcomposition may also comprise one or more surfactants and/or othercompounds found in conventional cleaning products as would be wellfamiliar to the skilled person.

Alternatively, the composition may be a coating composition adapted toprovide a C. diff. spore resistant layer on surfaces to which it isapplied. Accordingly, the composition typically comprises a solvent forsolubilising the one or more bile salts and the at least oneantimicrobial agents. The bile salt and antimicrobial agent aretypically as defined above. However, the composition may also comprisepolymerisable moieties or polymers which cause the coating compositionto set once it has been applied to a surface. Other optional additivesinclude leachable antimicrobial agents such as a silver ion source whichsecrete antimicrobial agents over time. Other additives, such ascolorants to improve the aesthetic of the coatings and dispersants andplasticisers to improve the “spray-ability” or improve the ease ofapplication of the coating composition to a surface may also be includedin the composition as would be appreciated by a person skilled in theart.

In a third aspect of the invention, there is provided a polymerobtainable by the polymerisation of a composition comprising one or morecompounds of general Formula 1, wherein the compounds comprise at leastone polymerisable moiety. Preferred aspects of Formula 1 may be asdefined above.

By polymerising one or more compounds according to Formula 1, it ispossible to fabricate a polymer having many repeating units, eachcomprising a bile salt moiety capable of stimulating germination of a C.diff. spore and an antimicrobial moiety capable of destroying the C.diff. bacterium as soon as it has germinated. This polymer can also bemanufactured into films and coating materials that can be incorporatedinto or on surfaces to provide a C. diff. spore killing property.

The position of the polymerisable moiety is not particularly restricted,provided that it does not interfere with the ability of the compound ofthe invention to at least partially promote germination of C. diff.spores and not prevent an antimicrobial activity. The polymerisablemoiety may form part of the X group on at least one of the compounds ofgeneral Formula 1. Alternatively, the polymerisable moiety may bepresent on the L group and/or M group on at least one of the compoundsof general Formula 1. It is typically the case that the L groupcomprises the polymerisable moiety.

As regards the structure of the polymerisable moiety, the skilled personwill appreciate that any moiety which can be polymerised withoutcompromising the spore germination and antimicrobial activity of thecompound of the invention can be used. Typically, wherein thepolymerisable group is unsaturated. Unsaturated functionalities, such ascarbon-carbon double bonds and triple bonds, provide a good “handle” forlinking together monomers using a number of known polymerisationtechniques with which the skilled person will be familiar.

Often the polymerisable group comprises an alkenyl group and it istypically the case that the polymerisable group comprises a terminalvinyl group.

The polymerisation used in the invention is typically a free radicalpolymerisation. The polymerisation technique is not particularly limitedbut may be any reasonable polymerisation method including for exampleliving polymerisations and living radical polymerisations. Examples ofsome polymerisation techniques which may be used include polymerisationsselected from: atom transfer radical polymerisation, reversibleaddition-fragmentation chain transfer polymerization, stable freeradical polymerization, living ionic polymerisation, living ring-openingmetathesis polymerization, group transfer polymerization, livingZiegler-Natta polymerization or combinations thereof.

Often the polymerisation is selected from: atom transfer radicalpolymerisation or reversible addition-fragmentation chain transferpolymerisation and it is most typically the case that the polymerisationis atom transfer radical polymerisation.

In another embodiment of the invention, there is provided a polymerwherein the composition comprises two or more compounds of generalFormula 1. The polymer need not be restricted to comprising oneparticular type of monomer according to general Formula 1 but may infact be a copolymer. Accordingly, a range of monomers according togeneral Formula 1 can be polymerised into the polymer which may, forexample, each comprise a different antimicrobial agent, bile salt and/orlinker.

Additionally, the composition (from which the polymer is obtained) mayfurther comprise one or more monomers other than the compounds ofgeneral Formula 1. The polymer need not solely consist of monomersaccording to general Formula 1 but may include other, typically moresimple monomers, in order to change the physical properties of thepolymer. For example, where a rigid antibacterial polymer is required,styrene could be incorporated into the polymer in order to modifyphysical properties of the polymer. Other modifications would beapparent to the skilled person in order to vary a range of properties ofthe polymer, such as melting point, acid/base resistance andhydrophilicity to name but a few. Further, cross linking agents may beintroduced i.e. monomers comprising moieties which can be made to reactwith other cross linking agents or complimentary groups in other polymerchains in order to form cross linked networks of polymers. Thefunctionality of the monomers not according to general Formula 1 mayalso be varied to provide particular chemical properties to the polymer.The monomer units may be selected from: amino acids, conjugated aminoacids, bile salts, antimicrobial agents or combinations thereof asdefined above.

As mentioned above the polymer may be a copolymer which may be a blockcopolymer, alternating copolymer or statistical copolymer or acombination thereof.

In a fourth aspect of the invention, there is provided a copolymerobtainable by the polymerisation of one or more first monomerscomprising a polymerisable group and an antimicrobial group; and one ormore second monomers comprising a polymerisable group and a bile saltgroup.

Typically the bile salt group and the antimicrobial groups havestructures as outlined above. The polymerisable groups present in thefirst and second monomers are not particularly limited nor are thesegroups required to be identical in both the first and second monomers.These groups need only be capable of undergoing polymerisation with oneanother to form a copolymer without removing either the C. diff. sporegerminating properties of the bile salt groups or the antimicrobialproperties of the antimicrobial groups.

The copolymer according to the fourth aspect of the invention also neednot solely consist of copolymers obtainable by the polymerisation of oneor more first monomers comprising a polymerisable group and anantimicrobial group; and one or more second monomers comprising apolymerisable group and a bile salt group. Additional monomers may beincluded, other than those having antimicrobial or bile salt functionalgroups. Such monomers can be included into the copolymer in order tochange the physical properties of the polymer in much the same way asoutlined above as would be clear to a person skilled in the art.

The chemical structure of the copolymer may also be varied byintroducing monomers having specific chemical functionality. Forinstance, the additional monomers may be selected from one of thenaturally occurring amino acids. Typically, the amino acids and/orconjugated amino acids may be selected from glycine, glutamine, leucine,isoleucine, alanine and taurine. It is typically the case that theadditional monomer is taurine.

Typically, the polymerisable group is unsaturated. Unsaturatedfunctionalities, such as carbon-carbon double bonds and triple bonds,provide a good “handle” on the first, second and additional monomerswhich can be linked together using a number of known polymerisationtechniques with which the skilled person will be familiar. Often thepolymerisable group comprises an alkenyl group and it is typically thecase that the polymerisable group comprises a terminal vinyl group.

It is also typically the case that the copolymer is a substantiallyalternating polymer. This ensures that the bile salt groups and theantimicrobial groups are in close proximity to one another along thepolymer chain in order to ensure that once germination is provoked bythe bile salt group, the antimicrobial group will be available todestroy the germinated C. diff. bacterium.

In an alternative embodiment, the copolymer of the fourth aspect of theinvention may be fabricated as a graft copolymer. A precursor polymermay be synthesised from one or more monomers comprising reactiveconnecting groups. The term “connecting group” is intended to refer to areactive moiety which can function as a point along the length of theprecursor polymer at which further functionality can be introduced.Typically, the connecting groups are suitable for undergoing clickchemistry, a technique known in the art, and the connecting group may beselected from either an alkyne or azide group. Alternatively, one ormore of the bile salt, antimicrobial agent and/or compound of formula 1(comprising both a bile salt group and an antimicrobial group) may befunctionalised with an alkene group which permits said compounds to begrafted onto a precursor polymer such as polyolefins. Examples ofpreferred polyolefins include polyethylene and in particular highdensity polyethylene (HDPE).

The precursor polymer may be reacted with antimicrobial agents and bilesalt compounds which have been functionalised with suitable groups(corresponding to the connecting groups present in the precursorpolymer) in order to form the copolymer according to the fourth aspectof the invention. The precursor polymer is typically obtainable by thepolymerisation of a first monomer comprising a first connecting groupand a second monomer comprising a second connecting group. By employingdifferent connecting groups, it is possible to more easily control theaddition of antimicrobial agents and bile salts on to the precursorpolymer backbone.

The polymerisation process used in the fourth aspect of the invention isnot particularly restricted, provided that the germinating properties ofthe bile salt group and antimicrobial properties of the antimicrobialgroup are not lost in the process, for example by degradation of saidgroups as a result of harsh reaction conditions or high temperatures.The polymerisation processes used is typically as defined above.

The antimicrobial groups and bile salt groups are typically as definedabove.

There is provided in a fifth aspect of the invention, a polymericmaterial which comprises either a bile salt and an antimicrobial agent;or a compound, polymer or copolymer according to the above aspects ofthe invention or combinations thereof. Where the polymeric materialcomprises a compound, polymer or copolymer or combinations thereofaccording to the above aspects of the invention, the polymeric materialmay additionally comprise a bile salt and/or an antimicrobial agent.

The polymeric material may be a conventional plastics material intowhich a bile salt and one or more antimicrobial agents (and/or compound,polymer, copolymer or combinations thereof according to the aboveaspects of the invention) are incorporated i.e. as a mixture.

By incorporating a bile salt and an antimicrobial agent intoconventional plastics, it is possible to imbue conventional plasticsmaterials with properties that cause C. diff. spores to germinate oncontact with the plastics material and then subsequently destroys thegerminated C. diff. spores.

Accordingly, in a first embodiment, the plastics material may beselected from any conventional plastics and it is typically the casethat the plastics are selected from: low density polyethylene (LDPE),high density polyethylene (HDPE), polypropylene (PP), polyvinyl chloride(PVC), polystyrene (PS), nylon, teflon (polytetrafluoroethylene) andthermoplastic polyurethanes (TPU).

The bile salts, antimicrobial agents, compounds, polymers and copolymersof the invention may be admixed into the polymers when they are in amolten state in order to homogeneously disperse these componentsthroughout the plastics material. Alternatively, said materials could beworked into the surface of the plastics material to form a coating onthe surface of the formed plastics material.

In addition, further additives may be combined with the plasticsmaterial to modify the plastics material's properties. For example, awide range of antibacterial, antiviral and antifungal agents may beincorporated into the plastics material and so too may other additivesfor modifying the flexibility, strength, temperature resistance andother physical properties of the plastic. Various additives, such ascolorants and plasticisers may be included in the plastics material aswould be familiar to persons skilled in the art.

In another embodiment, the plastics material is a polymer or copolymeraccording to the above aspects of the invention. In this embodiment, thepolymer itself rather than the additives incorporated therein, providesboth the C. diff. spore germinating properties and the antimicrobialproperties although these may be supplemented by incorporating bilesalts and/or antimicrobial agents and/or compounds of the invention intothe plastics material. Further optional additives may be included asdescribed above.

In a sixth aspect of the invention, there is provided an electronicdevice comprising a casing, wherein the casing comprises a compound orcomposition as described above. Typically, the casing is manufacturedfrom a polymeric material and/or has been coated with a coatingcomposition as described above.

Electronic devices, such a phones, watches, computers, tablets, andPersonal Digital Assistant are widespread among most populations and arein regular contact with human hands. As such, bacteria and othermicroorganisms are transferred to and from electronic devices on aregular basis. Such devices can be difficult to clean as many cleaningagents are water based and water can of course interfere with theelectric function of said devices.

Accordingly it is desirable to introduce antimicrobial agents into andon the surfaces of electronics devices to minimise spread of microbes.The term “casing” as used herein is intended to encompass the outersurface of an electronic device which typically contacts human skin innormal use. This includes screens, which may be glass or transparentplastics materials, as well as the housing (which contains the workingelectronics) as well as buttons which may be integral to the deviceand/or protrude from the device. Typically, the electronic devices aremobile phones or portable computer devices.

In a seventh aspect of the invention, there is provided a sheath ordetachable casing for an electronic device, wherein the sheath ordetachable sheath or detachable casing comprises a compound orcomposition as described above. Typically, the casing is manufacturedfrom a polymeric material and/or has been coated with a coatingcomposition as described above.

It is often the case that electronic devices are themselves housedwithin protective sheaths or detachable casings. This is usually toprotect the electronic devices from impacts in the event that the deviceis dropped or to provide an alternative aesthetic to the device.However, such sheaths or detachable casings then become a component onwhich microbes can be transferred. As such, it is desirable toincorporate the compounds, composition, polymers, copolymer and/orcombinations thereof as described above into these sheaths or detachablecasings in order to minimise the spread of microbes.

In an eighth aspect of the invention, there is provided a compoundaccording to the first aspect of the invention for use in the treatmentand/or prevention a disease selected from: C. difficile associateddisease (CDAD), and toxic megacolon.

In an ninth aspect of the invention, there is provided a method oftreating or preventing a disease selected from C. difficile associateddisease (CDAD), and toxic megacolon comprising administering a compoundaccording to the first aspect of the invention to a patient.

The invention also provides a compound as defined in the materials andmethods below, and its use in a method according to the invention.

The invention will be described with reference to the accompanyingfigures and examples.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows germination activity of HDPE (high densitypolyethylene)/“T101” (radical initiator 2,5-dimethyl-2,5-bis-(tert-butylperoxy)hexane) polymer films prepared with and without (control) C109(compound 129) at 3% (at 165° and 170° C.) and at 6% (at 160° C.).

FIG. 2 shows germination and antimicrobial efficacy of C109 (compound129) reactively processed in HDPE (high density polyethylene).

EXAMPLES

Clostridium difficile Spore Germination Potential of Compound 129

HDPE (high density polyethylene) samples containing the modified bilesalt compound 129, reactively processed under different compressionmoulding conditions, were tested to determine the germination activity.Polymer film samples containing 3% of compound 129 prepared (at molarratio of 0.005 of peroxide to bile salt) both at 165° C. and 170° C.demonstrated an equal level of germination activity at 2 hours,resulting in a 2.2 Log reduction compared to the HDPE/peroxide T101control (FIG. 1). However, after 24 and 46 hours, samples processed atthe lower temperature demonstrated a slightly higher level ofgermination activity suggesting that higher temperatures, used underthese conditions, give rise to a reduction in the germination activityof the modified bile salt compound 129. Samples prepared similarly butwith a higher amount of compound 129 (6%) did not perform well. Table 1shows that, following subsequent exposure to ethanol, compound 129prepared at 165° C./170° C. yields a 99.2% reduction in C. difficilespores. This confirms the germination activity of compound 129 whenincorporated into HDPE by reactive processing.

TABLE 1 Germination activity of compound 129 reactively processed withHDPE after subsequent exposure to ethanol Log Reduction % Reduction 24 224 Polymer 2 hours hours 96 hours hours hours 96 hours compound 1292.165 0.971 0.802 99.2 87.8 82.0 3% T101 170° C. compound 129 2.1651.598 1.154 99.2 97.1 92.0 3% T101 165° C. compound 129 0.752 0.5460.577 79.8 67.6 69.8 3% T101 160° C.Dual Activity of Compound 129 (Germination of C. difficile Spore andSubsequent Elimination)

Reactively processed HDPE polymer containing compound 129 is shown tohave the ability to both germinate the spores of C. difficile and reducethe subsequent metabolically active bio-load by 96.9%, see FIG. 2 andTable 2. This exemplifies, therefore, the dual action of compound 129and its efficacy both as a germinant and an antimicrobial. Reactiveprocessing the polymer at 170° C. changes markedly the dual activity ofcompound 129 giving a spore reduction of 79.6%.

TABLE 2 Germination and antimicrobial efficiency of compound 129 in HDPELog Reduction % Reduction 24 2 96 Polymer 2 hours hours 96 hours hours24 hours hours compound 129 0.747 0.666 0.998 79.6 75.4 88.5 3% T101170° C. compound 129 1.563 0.940 0.714 96.9 86.9 78.0 3% T101 165° C.compound 129 0.057 0.315 0.936 0.0 44.8 86.8 6% T101 160° C.Materials and Methods

Proton NMR spectra were obtained on a Bruker AC 250 instrument operatingat 250 MHz as solutions in CDCl₃ and referenced from δCDCl₃=7.26 ppmunless otherwise stated. Carbon NMR spectra were obtained on a Bruker AC250 instrument operating at 63 MHz as solutions in CDCl₃ and referencedfrom δCDCl₃=7.26 ppm unless otherwise stated. Infrared spectra wererecorded as KBr discs on a Mattson 3000 FTIR spectrophotometer.Electrospray mass spectrometry was carried out on a Waters LCT PremierToF (Time of flight) mass spectrometer. Electrospray mass spectrometryand accurate mass spectrometry was also carried out by the EPSRCNational Mass Spectrometry Facility in Swansea with a MAT95 magneticsector. Melting points were obtained using a Reichert-Jung Thermo Galanhot stage microscope and are corrected. All chemicals were purchasedfrom Sigma Aldrich.

Synthesis of methyl cholate (1)

Cholic acid (5.0 g, 12.2 mmol) was added to dry methanol (20 mL).Acetylchloride (0.5 mL, 0.04 mmol) was added under argon. The solutionwas heated at reflux for 45 minutes, then left to cool. Once at roomtemperature, the solution was cooled further on ice whereupon crystalsappeared. The solid material was collected by filtration and washed withsmall amounts of methanol to give methyl cholate as a white solid. Itwas dried at room temperature under vacuum.

Yield=3.548 g (68.9%)

Melting point=110.8-111.9° C.

¹H NMR (CDCl₃) δ ppm: 0.67 (s, 3H, Me-18), 0.88 (s, 3H, Me-19), 0.99 (d,3H, Me-21), 1.0-2.43 (m steroid structure) 2.49 (s, 1H, H—C—C═O), 3.47(s, 1H, H—C—OH), 3.66 (s, 1H, CH-3), 3.84 (d, 1H, CH-7), 3.95 (s, 1H,CH-12) ppm

¹³C NMR (CDCl₃) δ 174.77 (C═O), 73.26 (C12), 68.66 (C7), 52.67 (C3),51.50 (CH₃), 50.33, (47.05, 46.44, 41.82, 39.47, 39.01, 35.24, 34.70,31.06, 29.86, 28.17, 27.45, 26.60, 23.17 steroid structure) 22.44 (C19),17.31 (C21), 12.49 (C18).

MS (+APCI) m/z=482 (M⁺)

IR ν=3399 (OH), 2921, 2869, 1739 (C═O), 1449 cm⁻¹

Synthesis of cholic acid benzyl amide (2)

Methyl cholate (1 g, 2.3 mmol) was placed into a stainless steelpressure vessel along with benzyl amine (1.3 mL, 12.2 mmol) and toluene(5 mL). The pressure vessel was placed into an oil bath and heated to150° C. for 48 hours. The pressure vessel was left to cool beforedismantling it. The solution was poured into a round bottomed flask andplaced on ice. The solid product was then collected by filtration anddried at room temperature under vacuum. The material was recystallisedfrom ethanol/water to give a white, powdery solid which was dried atroom temperature under vacuum under vacuum.

Yield=0.378 g (37.8%)

Melting point=114.1-114.7° C.

¹H NMR (Methanol-d₄) δ ppm: 0.69 (s, 3H, Me-18), 0.91 (s, 3H, Me-19),1.02 (d, J=6.2 Hz, 3H, Me-21), 1.0-2.439 (m, steroid structure) 3.36 (m,1H, CH-3), 3.78 (s, 1H, H—C-7), 3.94 (s, 1H, CH-12), 4.35 (d, 2H, J=1.9Hz PH—CH₂), 7.29 (m, J=6.2 Hz, 6H, aromatic ring)

¹³C NMR (CDCl₃) δ 219.45, 218.76, 173.58 (C═O), 154.31 (aromatic ring),128.69 (aromatic ring), 127.85 (aromatic ring), 127.48 (aromatic ring),125.07 (aromatic ring), 76.51 (C12), (43.61, 41.92, 39.51, 35.26, 34.69steroid structure), 26.58 (C19), 17.44 (C21), 12.54 (C18)

MS (+APCI) m/z=Found 498.3578; calculated for C₃₁H₄₈N₁O₄ 497.71; −1.4ppm

IR ν=3411, 2917, 1644 (C═O), 1540, 1457 cm⁻¹

Synthesis of N-(4-aminobutyl) cholanamide (3)

Methyl cholate (1 g, 2.3 mmol) along with 1,4-diaminobutane (1,4-DAB)(10 mL) was put into a stainless steel pressure vessel and tightlysecured. It was placed into an oil bath and heated to 150° C. for 48hours. The pressure vessel was left to cool to room temperature beforedismantling it. 30 mL chloroform was added to dissolve the product andexcess 1,4-DAB which was then poured into a round bottomed flask. Thechloroform was taken off using a rotary evaporator and the 1,4-DAB wastaken off under reduced pressure rotary evaporation. Dichloromethane (90mL) was added to precipitate the product. The yellow solid was collectedby filtration and dried at room temperature under vacuum.

Yield: 0.3 g, (30.14%)

Melting point: 116.4-124.5° C.

¹H NMR (CDCl₃) δ ppm: 0.68 (s, 3H, Me-18) 0.89 (s, 3H, Me-19) 1.00 (d,J=6.3 Hz, 3H, Me-21) 1.0-2.43 (m, steroid structure) 1.22 (dd, J-14.1,8.5 Hz, CH) 2.71 (s, CH₂) 3.28 (m, 1H, CH-3) 3.86 (s, 1H, CH-7) 3.98 (s,1H, CH-12)

¹³C NMR (DMSO) δ 172.43 (C═O), 107.85, 70.97 (C12), 66.20 (C7), (46.08,45.70, 41.35, 40.17, 39.84, 37.97, 35.65, 35.13, 34.87, 34.36, 28.55,27.51, 27.25, 26.42, 26.20, 22.81, 22.60 steroid ring), 41.71 (CH₂),40.51 (CH₂), 30.38 (CH₂), 20.75 (C19), 19.52 (C21), 17.10, 12.31 (C18).

MS (+APCI) m/z=Found 479.3849; calculated for C₂₈H₅₀N₂O₄ 478.3771; −1.0ppm

IR (KBr) ν=3274, 2935, 2898, 2865, 2831, 1736, 1669 (C═O), 1548 cm⁻¹

Synthesis of N-[2-(2-aminomethylamino)ethyl] cholanamide (4)

Methyl cholate (0.5 g, 1.15 mmol) along with diethylenetriamine (2 mL)was put into a round bottomed flask under argon. It was placed into anoil bath and heated to 95° C. for 48 hours. The flask was left to coolto room temperature. Aceonitrile (15 mL) was added to precipitate theproduct which was then collected by filtration. Solvent extractionbetween water and chloroform to carried out to purify the product. Thechloroform was evaporated under reduced pressure. The solid was dried atroom temperature under vacuum at room temperature under vacuum.

Yield=0.07 g (14%)

Melting point: 205.6-206.8° C.

¹H NMR (CDCl₃) δ ppm: 0.66 (s, 3H, Me-18) 0.87 (s, 3H, Me-19) 0.9 (d,J=6.0 Hz, 3H, Me-21) 1.0-2.44 (m, steroid structure) 2.69 (d, J=5.9 Hz,CH₂) 2.75-2.88 (m) 3.35 (s) 3.47 (m, 1H, CH-3) 3.57 3.69 3.72 3.83 (s,1H, CH-7) 3.94 (s, 1H, CH-12) 7.01 (s, 1H, NH)

¹³C NMR (DMSO) δ ppm: 172.59 (C═O), 70.98 (C12), 70.41 (C3), 66.22 (C7),(45.70, 41.50, 41.32, 40.49, 40.16, 35.28, 34.35, 32.44, 31.57, 30.38,28.53, 27.28, 26.18 steroid ring), 22.58 (C19), 17.08 (C21), 12.32(C18).

MS (+APCI) m/z=Found 494.3958; calculated for C₂₉H₅₃N₃O₄ 493.3880; 0.6ppm

IR (KBr) ν=3253, 2929, 2865, 1751, 1648 (C═O), 1557 cm⁻¹

Synthesis of N-[3-(cyclohexylamino)propyl] cholanamide (5)

Methyl cholate (2 g, 4.6 mmol) along with cyclohexyl-1,3-propanediamine(4.08 mL) was added to a round bottomed flask under nitrogen. The flaskplaced in a oil bath at 95° C. for 48 hours before raising thetemperature to 120° C. for 12 hours. The flask was allowed to coolbefore chloroform (30 mL) was added to dissolve the product. Solventextraction between chloroform (30 mL) and water (30 mL) removed anyexcess amine. This was repeated three times. The chloroform layer wasdried over magnesium sulphate and evaporated under reduced pressure togive a solid that was recrystallized from chloroform-petrol 60-80.

Yield=1.2 g (60%)

Melting point: 104.5-105.8° C.

¹H NMR (CDCl₃) δ ppm: 0.67 (s, 2H, Me-18) 0.89 (s, 2H, Me-19) 0.98 (d,3H, Me-21) 1.0-2.43 (m, steroid structure) 2.61-2.79 (m, ring) 3.26 (m,1H, CH-3) 3.43 (m, 1H, NH) 3.84 (s, 1H, CH-7) 3.97 (s, 1H, CH-12)

¹³C NMR (CDCl₃) δ ppm: 173.94 (C═O), 71.85 (C12), 68.27 (C7), 56.87(CH), (46.51, 41.49, 40.08, 39.58, 38.83, 35.38, 31.74, 31.62, 30.41,29.08, 28.17, 27.61, 26.43 (CH), 26.10 steroid ring), 45.30 (CH₂), 40.68(CH₂), 25.11 (CH), 34.80 (CH), 33.12 (CH), 23.31, 22.69, 22.51 (C19),17.57 (C21), 14.17, 12.50 (C18), 11.48.

MS (+APCI) m/z=Found 547.4475; calculated for C₃₃H₅₈N₂O₄ 546.4397; 0.4ppm

IR (KBr) ν=3265, 3068, 2919, 2853, 1648 (C═O), 1554 cm⁻¹

Coupling of Cholic Acid with Ethylchloroformate and Various Amines

General procedure for the coupling of cholic acid to primary andsecondary amines Cholic acid (0.5 g, 1.2 mmol) was dissolved in THF (30mL) along with triethylamine (2.9 mL, 0.3 mmol). The solution was put onice for 10 minutes before ethylchloroformate (0.13 mL, 0.013 mmol) wasdripped in over 10 minutes. The solution was allowed to react for twohours at room temperature. The required amine (1.2 mmol) was added andleft to react for 3 hours. The reaction was quenched with water (30 mL).The mixture was washed with water (3×30 mL). The organic layer was driedover magnesium sulphate and the solvent was evaporated under reducedpressure. Solvent extraction between water and ethyl acetate waspreformed 3 times before the organic layer was removed on the rotaryevaporator. The product was dried at room temperature under vacuum.

Synthesis of N-[3-(cyclohexylamino)propyl] cholanamide (6)

The procedure was followed as the above with the exception of theremoval of the two hour wait before adding the amine.Cyclohexylpropanediamine (0.24 mL, 1.56 mmol) added in one portion. Thecrude material was purified by flash chromatography eluting withmethanol.

Yield=0.36 g (72%)

Melting point: 104.5-105.9° C.

¹H NMR (CDCl₃) δ ppm: 0.67 (s, 2H, Me-18) 0.89 (s, 2H, Me-19) 0.98 (d,3H, Me-21) 1.0-2.439 (m, steroid structure) 2.61-2.79 (m, ring) 3.26 (m,1H, CH-3) 3.43 (m, 1H, NH) 3.84 (s, 1H, CH-7) 3.97 (s, 1H, CH-12)

¹³C NMR (CDCl₃) δ ppm: 173.94 (C═O), 73.08 (12), 71.85 (C3), 68.27 (C7),56.87 (CH), (46.51, 41.49, 40.08, 39.58, 38.83, 35.38, 31.74, 31.62,30.41, 29.08, 28.17, 27.61, 26.10 steroid ring), 45.30 (CH₂), 40.68(CH₂), 34.80 (CH₂), 33.12 (CH₂), 26.43 (CH), 25.11 (CH), 23.31, 22.69,22.51 (C19), 17.57 (C21), 14.17, 12.50 (C18), 11.48.

MS (+APCI) m/z=Found 547.4461; calculated for C₃₃H₅₉N₂O₄ 547.4469; −1.5ppm

IR (KBr) ν=3265, 3068, 2919, 2853, 1648 (C═O), 1554 cm⁻¹

Synthesis of N-[3-(dibutylamino)propyl] cholanamide (7)

The procedure was followed as above with 3-dibutylaminopropylamine (0.22mL, 1.18 mmol). Purification of the crude product was done dissolvingthe product in ethyl acetate (10 mL), removing solid impurities byfiltration followed by removing the organic solvent. The product wasdried at room temperature under vacuum.

Yield=0.42 g (84%)

Melting point: 145.1-145.9° C.

¹H NMR (CDCl₃) δ ppm: 0.68 (s, 3H, Me-18) 0.89 (s, 3H, Me-19) 0.92 (d,J=6.3 Hz 3H, Me-21) 1.0-2.439 (m, steroid structure) 2.44 (m, butylchain) 2.55 (m, butyl chain) 3.33 (m, propyl chain) 3.44 (m, 1H, CH-3)3.84 (s, 1H, CH-7) 3.97 (s, 1H, CH-12) 7.43 (m, 1H, NH)

Carbon=N/A

MS (+APCI) m/z=577.4931

IR (KBr) ν=3299, 3089, 2919, 2853, 2358, 1727, 1642 (C═O), 1545, 1463cm⁻¹

Synthesis of N-(2-pyrrolidin-1-ylethyl) cholanamide (10)

The procedure was followed as above with 1-(2-aminoethyl) pyrrolidine(0.21 mL, 1.8 mmol).

Yield=0.74 g (74%)

Melting point: 96-97.1° C.

¹H NMR (CDCl₃) δ ppm: 0.68 (s, 3H, Me-18) 0.89 (s, 3H, Me-19) 1.00 (d,J=6.2 Hz, 3H, Me-21) 1.0-2.43 (m, steroid structure) 2.23 (d, J=12.1 Hz,CH₂) 2.64 (s 2H, CH₂) 3.44 (m 1H, CH-3) 3.84 (s, 1H, CH-7) 3.96 (s, 1H,CH-12) 6.40 (s, 1H, NH)

¹³C NMR (DMSO) δ ppm: 172.45 (C═O), 79.14, 70.98 (C12), 66.21 (C7),55.02 (CH₂), 53.58 (CH₂ ring), (46.12, 45.70, 41.49, 37.81, 35.13,34.35, 32.51, 31.70, 26.18 steroid ring), 41.34 (CH₂) 23.08 (CH₂ ring),22.59 (C19), 17.07 (C21), 12.30 (C18).

MS (+APCI) m/z=Found 505.3993; calculated for C₃₀H₅₃N₂O₄ 505.4000; −1.4ppm

IR (KBr) ν=3287, 2929, 2862, 2155, 1642 (C═O) cm⁻¹

Synthesis of N-[3-(dimethylamino)propyl] cholanamide (12)

The procedure was followed as above with 3-(dimethylamino)-1-propylamine(0.18 mL, 1.76 mmol).

Yield=0.70 g (70%)

Melting point: 163.2-164.0° C.

¹H NMR (CDCl₃) δ ppm: 0.67 (s, 2H, Me-18) 0.87 (s, 2H, Me-19) 0.97 (d,J=6.2 Hz, 3H, Me-21) 1.0-2.43 (m, steroid structure) 2.21 (m, CH₂) 2.33(s, 6H, 2CH₃) 3.30 (m, J=5.7 Hz, 1H) 3.82 (s, 1H, CH-7) 3.96 (s, 1H,CH-12) 6.97 (s, 1H, NH)

¹³C NMR (DMSO) δ ppm: 176.07 (C═O), 70.99 (C12), 70.41 (C3), 66.23 (C7),56.42 (CH₃), (46.25, 44.96, 41.50, 41.31, 40.14, 35.25, 34.84, 34.36,32.61, 30.38, 28.51, 27.30, 26.77, 26.17 steroid ring), 45.72 (CH₃),40.47 (CH₂), 31.52 (CH₂), 22.80, 22.58 (C19), 17.04 (C21), 12.34 (C18).

MS (+APCI) m/z=Found 493.3994; calculated for C₂₉H₅₃N₂O₄ 493.4000; −1.2ppm

IR (KBr) ν=3387, 2932, 2862, 2209, 1991 cm⁻¹

Synthesis of N-(1-phenyl-4-piperidyl) cholanamide (13)

The procedure was followed as above with 4-amino-1-benzylpiperidine(0.34 mL, 1.79 mmol).

Yield=0.88 g (88%)

Melting point: 79.9-86.5° C.

¹H NMR (CDCl₃) δ ppm: 0.67 (s, 3H, Me-18) 0.89 (s 3H, Me-19) 0.99 (d,J=6.1 Hz, 3H, Me-21) 1.0-2.43 (m, steroid structure) 2.83 (d, J=11.9 Hz)3.43 (m, 1H, CH-3) 3.48 (d, J=3.2 Hz) 3.83 (s, 1H, CH-7) 3.96 (s, 1H,CH-12) 5.72 (d, J=8.0 Hz, 1H) 7.317 (s, 1H, NH)

¹³C NMR (DMSO) δ ppm: 171.78 (C═O), 138.72, 138.63, (128.65, 128.09,126.77 aromatic ring), 70.98 (C12), 70.41 (C3), 66.21 (C7), 62.15,51.97, 51.87, (46.12, 45.71, 41.50, 41.33, 40.51, 40.32, 40.17, 35.29,35.11, 34.85, 34.36, 32.59, 31.73, 31.62, 30.39, 28.53, 22.77 steroidring), 27.27 (CH₂ ring), 26.19 (CH₂ ring) 22.59 (C19), 17.12 (C21),14.63, 12.31 (C18).

MS (+APCI) m/z=Found 519.4138; calculated for C₃₆H₅₇N₂O₄ 581.4313.−61=benzene ring

IR (KBr) ν=3279, 3006, 2925, 2810, 2358, 2162, 1782, 1646 (C═O), 1513cm⁻¹

Synthesis of 1-(3-phenylimidazolidin-1-yl) cholanone (14)

The procedure was followed as above with 1-(4-pyridyl)piperazine (0.24g, 1.5 mmol).

Yield=0.523 g (53%)

Melting point: 139.6-141.2° C.

¹H NMR (CDCl₃) δ ppm: 0.68 (s, 3H, Me-18) 0.89 (s, 3H, Me-19) 0.98 (d,3H, Me-21) 1.0-2.43 (m steroid structure) 2.26 (m 1H) 3.63 (m 1H CH-3)3.85 (m 1H CH-7) 3.98 (s 1H CH-12) 4.12 (d, J=6.95 Hz, 2H) 6.55-6.81 (m,2H NH) 8.12-8.45 (m, 2H NH)

¹³C NMR (DMSO) δ ppm: 173.30 (C═O), 171.33, 154.17, 149.80, 108.29,99.48, 71.02 (C12), 70.41 (C3), 66.21 (C7), 59.58, (45.74, 45.74, 45.12,40.47, 40.14, 35.27, 34.96, 34.35, 30.70, 30.37, 28.49, 26.17 steroidrings), 27.27 (CH₂ ring) 22.77, 22.59 (C19), 17.17, 16.87 (C21), 14.11,12.28 (C18).

MS (+APCI) m/z=Found 454.3528; calculated for C₃₃H₅₁N₃O₄ 553.387.MI—N(Ph)-NCH₂CH₂

IR (KBr) ν=3396, 3250, 2929, 2859, 2158, 1724, 1593, 1515 cm⁻¹

Synthesis of 1-[4-(2-hydroxyethyl)piperazin-1-yl] cholanone (15)

The procedure was followed as above with 1-(2-hydroxyethyl)piperazine(0.2 mL, 1.5 mmol). Proton NMR analysis showed excess of amine present.

Yield=0.4 g (80%)

Melting point: 167.8-168.1° C.

¹H NMR (CDCl₃) δ ppm: 0.70 (s, 2H, Me-18) 0.90 (s, 2H, Me-19) 1.10 (d,J=5.3 Hz, 3H, Me-21) 1.0-2.43 (m, steroid structure) 2.50-2.63 (m, CH—N)3.48 (m, J=4.9 1 Hz, CH-3 and CH) 3.64 (d, J=5.3 Hz, CH—N) 3.86 (s, 1H,CH-7) 3.98 (s, 1H, CH-12)

¹³C NMR (DMSO) δ ppm: 170.97 (C═O), 154.56, 71.00 (C12), 70.40 (C3),66.22 (C7), 60.61, 60.12 (CH₂), 58.43 (CH₂), 52.93 (CH₂ ring), 52.80,(46.08, 43.29, 41.48, 35.26, 35.21, 34.83, 34.35, 31.19, 30.39, 29.48,28.47, 27.29, 26.17 steroid ring), 45.74 (CH₂ ring), 22.81, 22.59 (C19),17.14 (C21), 14.54, 12.32 (C18).

MS (+APCI) m/z=521.3943

IR (KBr) ν=3617, 3414, 2916, 2853, 2810, 1687 (C═O), 1624 cm⁻¹

Synthesis of N-octadecylcholanamide (16)

The procedure was followed as above with octadecylamine (0.8 g, 2.9mmol). Further purification carried out using solvent extraction (×3)between THF/water (30 mL) and chloroform (30 mL). The organic layer wasdried over magnesium sulphate and the solvent was evaporated underreduced pressure. The product was dried at room temperature undervacuum.

Yield=0.922 g (90%)

Melting point: 83.6-84.4° C.

¹H NMR (CDCl₃) δ ppm: 0.66 (s, 3H, Me-18) 0.87 (d, J=3.4 Hz, 3H, Me-19)0.97 (d, J=5.9 Hz, 3H, Me-21) 1.0-2.43 (m, steroid structure) 1.23 (s,37H, CH₂) 3.2 (q, J=7.3, 6.8 Hz) 3.43 (m, 1H, CH-3) 3.83 (s, 1H, CH-7)3.96 (s, 1H, CH-12) 5.56 (s, 1H, NH)

¹³C NMR (DMSO) δ ppm: 172.90 (C═O), 172.43, 70.98 (C12), 70.40 (C3),66.21 (C7), 56.68, (46.15, 45.68, 45.08, 41.48, 36.68, 35.12, 34.35,31.75, 31.26, 30.34, 29.02, 28.67, 27.13, 26.18 steroid ring), 22.79(CH₂), 22.59 (CH₂), 22.57 (C19), 22.06, 17.08 (C21), 13.92 (CH₂), 12.31(C18).

MS (+APCI) m/z=Found 660.5923; calculated for C₄₂H₇₇N₁O₄ 660.5925; −0.4ppm

IR (KBr) ν=3299, 2916, 2847, 1648 (C═O), 1642 cm⁻¹

Synthesis of N-benzylcholanamide (17)

The procedure was followed as above with benzylamine (0.12 mL, 1.1mmol). The product was recrystallised in dichoromethane.

Yield=0.282 g (56.4%)

Melting point=114.1-114.7° C.

¹H NMR (Methanol-d₄) δ ppm: 0.69 (s, 2H, Me-18), 0.91 (s, 2H, Me-19),1.02 (d, J=6.2 Hz, 3H, Me-21), 1.0-2.43 (m, steroid structure) 3.36 (m,1H, CH-3), 3.78 (s, 1H, H—C-7), 3.94 (s, 1H, CH-12), 4.35 (d), 7.29 (m,J=6.2 Hz, 6H)

¹³C NMR (DMSO) δ ppm: 172.55 (C═O), (139.77, 128.17, 127.06, 126.60aromatic ring), 70.97 (C12), 70.41 (C3), 66.20 (C7), (46.15, 45.71,41.89, 40.49, 40.16, 35.28, 35.09, 34.86, 34.36, 34.36, 32.49, 31.77,30.38, 28.53, 27.30, 26.18 steroid ring), 22.61 (C19), 17.06 (C21),12.31 (C18).

MS (+APCI) m/z=Found 498.3569; calculated for C₃₁H₄₇N₁O₄ 498.3578; −1.8ppm

IR ν=3411, 2917, 1644, 1540, 1457 cm⁻¹

Synthesis of 2-cholanamidobenzamide (18)

The procedure was followed as above with 2-aminobenzomide (0.5 g, 3.7mmol). Further separation of the crude product to purify it was carriedout with sodium hydrogen carbonate (30 mL) and 2M hydrochloric acid (30mL). The organic layer was dried over magnesium sulphate and the solventwas evaporated under reduced pressure. The product was dried at roomtemperature under vacuum.

Melting point: 103-104° C.

¹H NMR (CDCl₃) δ ppm: 0.69 (s, 3H, Me-18) 0.89 (s, 3H, Me-19) 1.03 (d,J=6.0 Hz, 3H, Me-21) 1.0-2.43 (m steroid structure) 2.10 (s) 3.47 (m,J=7.9 Hz, 1H, CH-3) 3.84 (s, 1H, CH-7) 3.98 (s, 1H, CH-12) 6.25 7.07 (m,ring) 7.52 (d, J=8.3 Hz, ring) 8.62 (d, J=8.4 Hz, 1H, NH) 11.10 (s)

¹³C NMR (CDCl₃) δ ppm: 176.74, 176.03 (C═O), (145.05, 137.37, 133.74,124.55 aromatic ring), 71.47 (C12), 51.28, 51.00, (46.75, 46.58, 45.75,45.42, 45.09, 44.75, 44.42, 44.09, 43.75, 40.29, 39.61, 31.43, 27.84steroid ring), 22.27 (C19), 17.56 (C21).

MS (+APCI) m/z=Found 527.3473; calculated for C₃₁H₄₆N₅O₅ 527.3479; −1.2ppm

IR (KBr) ν=3350, 3220, 2932, 2865, 1721, 1660 (c=C═O), 1612, 1581 cm⁻¹

Synthesis of N-(4-benzoylphenyl)cholanamide (19)

The procedure was followed as above with 4-aminobenzophenone (0.23 g,1.17 mmol).

Yield=0.1270 g (25.4%)

Melting point: 125.8-127.6° C.

¹H NMR (CDCl₃) δ ppm: 0.70 (s, 3H, Me-18) 0.90 (s, 2H, Me-19) 1.02 (d,J=6.1 Hz, 3H, Me-21) 1.0-2.43 (m, steroid structure) 3.48 (m, 1H, CH-3)3.86 (s, 1H, CH-7) 3.99 (s, 1H, CH-12) 7.37-7.65 (m, 6H, ring) 7.65-7.91(m, 6H, ring) 8.25 (s, 1N, NH)

¹³C NMR (DMSO) δ ppm: 194.48 (C═O), 172.42 (C═O), (143.55, 137.59,132.14, 131.11, 130.99, 129.30, 128.43, 118.16 aromatic rings), 66.21(C7), (45.72, 40.50, 40.17, 39.84, 39.50, 39.17, 38.84, 38.50, 35.27,35.12, 34.36, 34.36, 33.56, 31.29, 30.39, 28.54, 27.31, 26.20 steroidring), 22.60 (C19), 17.13 (C21), 12.34 (C18).

MS (+APCI) m/z=Found 588.3684; calculated for C₃₇H₅₀N₁O₅ 587.36; −0.6ppm

IR (KBr) ν=3317, 3098, 2932, 2865, 1967, 1645, 1584, 1521 cm⁻¹

Synthesis of N-(4-vinylphenyl)acetamide (20)

The procedure was followed as above with 4-vinyl aniline (0.42 mL, 3.5mmol). Further purification with washing product dissolved in ethylacetate (30 mL) with 2M HCl (30 mL) four times. The organic layer wasdried over magnesium sulphate and the solvent was evaporated underreduced pressure. The product was dried at room temperature undervacuum. Proton NMR analysis showed the product contained excess 4-vinylaniline.

Melting point: N/A

¹H NMR (CDCl₃) δ ppm: 0.69 (s, 3H, Me-18) 0.90 (s, 3H, Me-19) 1.02 (d,J=6.0 Hz, 3H, Me-21) 1.0-2.43 (m, steroid structure) 2.36 (s) 2.62 (s)3.47 (m, 1H, CH-3OH) 3.87 (s, 1H, CH-7) 3.99 (s, 1H, CH-12) 5.18 (d,J=11.5 Hz, 2H, CH₂) 5.67 (d, J=18.0 Hz, 2H, CH₂) 6.61 (dd) 7.36 (d,J=5.4 Hz, aromatic) 7.52 (d, J=8.5 Hz, aromatic)

¹³C NMR (DMSO) δ 171.74 (C═O), (153.40, 139.14, 138.93, 131.73, 131.22,128.96, 128.86, 128.17, 126.58, 126.47, 125.28, 118.85, 117.95, 112.28aromatic ring, with excess 4-vinyl aniline), (137.30, 136.16, 112.46C═C), 70.95 (C12), 70.40 (C3), 66.19 (C7), 60.14, (46.06, 45.69, 41.46,41.34, 40.42, 40.09, 35.27, 35.16, 34.85, 34.35, 33.43, 31.43, 30.35,28.53, 27.28, 26.16 steroid ring), 22.78, 22.59 (C19), 21.02, 17.11(C21), 14.48, 12.33 (C18), 10.94.

MS (+APCI) m/z=Found 510.3570; calculated for C₃₂H₄₈N₁O₄ 510.3578; −1.5ppm

IR (KBr) ν=3429, 3296, 3101, 3044, 2929, 2868, 2364, 1672 (C═O), 1587,1521, 1508, 1460 cm⁻¹

Synthesis of N-(2-dimethylaminoethyl)cholamide (21)

The procedure was followed as above with cholic acid (5 g, 12.2 mmol),triethylamine (2.9 mL, 28.7 mmol), ethylchloroformate (1.3 mL, 12 mmol)and dimethylethylenediamine (1.3 mL, 14.7 mmol).

Yield=3.2185 g (64%)

Melting point: 181-182.2° C.

¹H NMR (CDCl₃) δ ppm: 0.66 (s, 3H, Me-18) 0.87 (s, 3H, Me-19) 0.98 (d,J=6.0 Hz, 3H, Me-21) 1.0-2.43 (m steroid structure) 2.37 (s, 4H, 2CH₂)2.62 (t, 2H, CH₂) 3.39 (d, J=5.7 Hz, CH₂ plus CH-3) 3.81 (s, 1H, CH-7)3.94 (s, 1H, CH-12) 7.17 (s, 1H, NH)

¹³C NMR (CDCl₃) δ ppm: 218.02, 178.14, 174.59 (C═O), 73.01, 71.80 (C12),68.37 (C7), 57.82 (CH₂), (46.54, 46.49, 44.98, 44.63, 41.69, 39.73,39.59, 35.62, 35.49, 34.87, 33.15, 31.75, 30.62, 28.30, 27.70, 26.42steroid ring), 36.26 (CH₃), 23.39, 23.32, 22.58 (C19), 17.55 (C21),14.73, 12.53 (C18)

MS (+APCI) m/z=Found 479.3835; calculated for C₂₈H₅₀N₂O₄ 479.3843 −1.7ppm

IR (KBr) ν=3484, 3250, 3074, 2925, 2865, 1715, 1633, 1551 cm⁻¹

Each cholic acid derivative was dissolved in either chloroform ordichloromethane along with a tenfold excess of the alkylating agent. Themixtures were left at room temperature for 4-48 hours. Upon theprecipitation of the product, the solid was collected by filtration andwashed with solvent, dried and purified if necessary.

Synthesis of 3-cholanamidopropyl(trimethyl)ammonium iodide (24)

N-[3-(dimethylamino)propyl] cholanamide (12) (0.5 g, 1.0 mmol) wasdissolved in DCM (25 mL) along with methyl iodide (1.4 mL, 4.3 mmol).The reaction was left for 48 hours where upon a yellow solid formed. Thesolid was collected by filtration and washed with chloroform (10 mL)before drying at room temperature under vacuum.

Yield=0.18 g (36%)

Melting point: 134.6-135.4° C.

¹H NMR (D₂O) δ ppm: 0.70 (s, 3H, Me-18) 0.90 (s, 3H, Me-19) 0.96 (d,J=Hz, 3H, Me-21) 1.0-2.43 (m, steroid structure) 3.11 (s, 9H, 3CH₃) 3.30(ddt, J=17.8 Hz) 3.48 (dd, J=10.4, 5.3 Hz, 1H, CH-3) 3.89 (s, 1H, CH-7)4.05 (s, 1H, CH-12) 7.65 (s, 1H, NH)

¹³C NMR (DMSO) δ ppm: 212.03, 172.84 (C═O), 79.97, 70.95 (C12), 70.37(C3), 66.20 (C7), 52.27 (CH₃), 52.18, 45.68 (CH₂), 40.52 (CH₂), (40.43,40.18, 40.10, 35.27, 34.35, 32.33, 29.95 steroid ring), 22.97, 22.60(19), 17.11 (C21), 12.31 (C18), 6.83.

MS (+APCI) m/z=Found 507.4150; calculated for C₃₀H₅₅N₂O₄ 507.4156; −1.3ppm

IR (KBr) ν=3387, 2929, 2862, 1700, 1642 cm⁻¹

Synthesis of 3-cholanamidopropyl-ethyl-dimethyl-ammonium iodide (25)

N-[3-(dimethylamino)propyl] cholanamide (12) (0.5 g, 1.0 mmol) wasdissolved in DCM (20 mL) along with ethyl iodide (0.4 mL, 1.3 mmol) andmethanol (1 mL). The reaction was left for 1 week before the solvent wasremoved under reduced pressure. The solid was washed with ether (10 mL)and chloroform (10 mL) before drying. The product was a yellow solid.

Yield=0.267 g (53%)

Melting point: 122.5-123.1° C.

¹H NMR (D₂O) δ ppm: 0.72 (s, 3H, Me-18) 0.92 (s, 3H, Me-19) 0.97 (d, 3H,Me-21) 1.0-2.43 (m, steroid structure) 3.04 (s, 6H, CH₂) 3.21-3.34 (m,4H, 2CH₂) 3.38 (q, J=7.3 Hz, 2H, CH₂) 3.44-3.62 (m, 1H, CH-3) 3.89 (s,1H, CH-7) 4.06 (s, 1H, CH-12) 7.67 (s, 1H, NH)

¹³C NMR (DMSO) δ ppm: 197.83, 172.88 (C═O), 70.96 (C12), 70.37 (C3),66.20 (C7), 60.46, 58.59 (CH₃), 49.59, 49.52, (45.96, 41.46, 41.38,40.51, 40.17, 39.84, 39.51, 39.42, 39.17, 39.01, 38.84, 38.51, 35.47,35.25, 35.15, 34.87, 34.34, 32.35, 30.35, 28.55, 27.26, 26.20 steroidring), 45.68 (CH₂), 31.52 (CH₂), 22.77, 22.59 (C19), 22.52 (CH₂), 17.10(C21), 12.31 (C18), 7.76.

MS (+APCI) m/z=Found 521.4305; calculated for C₃₁H₅₇N₂O₄ 521.4313; −1.5ppm

IR (KBr) ν=3420, 3256, 2913, 2856, 2243, 1639 (C═O) cm⁻¹

Synthesis of 3-cholanamidopropyl-propyl-dimethyl-ammonium iodide (26)

N-[3-(dimethylamino)propyl] cholanamide (12) (0.5 g, 1.0 mmol) wasdissolved in DCM (20 mL) along with 1-iodopropane (1.5 mL, 5.0 mmol) andmethanol (1 mL). The reaction was left for 1 week before solventextraction (×3) between dichloromethane (20 mL) and water (20 mL). Theorganic layer was dried with magnesium sulphate before being removedunder reduced pressure. The product was a white solid which was dried atroom temperature under vacuum.

Yield=0.233 g (46%)

Melting point: 108.1-108.9° C.

¹H NMR (DMSO) δ ppm: 0.57 (s, 3H, Me-18) 0.79 (s, 3H, Me-19) 0.86-0.94(m, 37H, Me-21 plus 2CH₂) 1.0-2.43 (m, steroid structure) 2.94-3.28 (m,14H) 3.61 (s, 1H, 7CH) 3.77 (s, 1H, 12CH) 3.99 (d, J=, 1H 3OH) 4.10 (d,J=, 1H, 7OH) 4.32 (d, J=4.0 Hz, 1H, 120H) 7.88 (t, J=5.7 Hz, NH)

¹³C NMR (DMSO) δ ppm: 172.92 (C═O), 70.97 (C12), 70.37 (C3), 66.20 (C7),64.37, 61.10, 50.20, 50.16, (45.97, 35.45, 35.15, 34.86, 34.34, 32.37,30.34, 28.53, 26.20 steroid ring), 45.68 (CH₃), 31.54 (CH₂), 27.26(CH₂), 22.58 (C19), 17.10 (C21), 15.32 (CH₃), 12.31 (C18), 10.45 (CH₂).

MS (+APCI) m/z=Found 535.4461; calculated for C₃₂H₅₉N₂O₄ 535.4469; −1.6ppm

IR (KBr) ν=3396, 2935, 2865, 2246, 2124, 1706, 1645 (C═O), 1533 cm⁻¹

Synthesis of 3-cholanamidopropyl-butyl-dimethyl-ammonium iodide (27)

N-[3-(dimethylamino)propyl] cholanamide (12) (0.5 g, 1.0 mmol) wasdissolved in DCM (20 mL) along with 1-iodobutane (1.2 mL, 4 mmol) andmethanol (1 mL). The reaction was left for 1 week before solventextraction (×3) between dichloromethane (20 mL) and water (20 mL). Theorganic layer was dried with magnesium sulphate before being removedunder reduced pressure. The product was a white solid which was dried atroom temperature under vacuum.

Yield=0.3115 g (62%)

Melting point: 125.8-126.4° C.

¹H NMR (D₂O) δ ppm: 0.68 (s, 3H, Me-18) 0.88 (s, H, Me-19) 0.84-1.00 (m,5H, Me-21 plus CH₂) 1.0-2.43 (m, steroid structure) 3.02 (s, 6H, CH₂)3.14-3.32 (m, 6H, CH₂) 3.47 (m, 1H, 3CH) 3.86 (s, 1H, 7CH) 4.02 (s, 1H,12CH)

MS (+APCI) m/z=Found 549.4619; calculated for C₃₃H₆₁N₂O₄ 549.4626; −1.2ppm

IR (KBr) ν=3378, 2932, 2865, 2358, 2337, 2155, 2009, 1976, 1651, 1633(C═O), 1539 cm⁻¹

Synthesis of 3-cholanamidopropyl-pentyl-dimethyl-ammonium iodide (28)

N-[3-(dimethylamino)propyl] cholanamide (12) (0.5 g, 1 mmol) wasdissolved in chloroform (75 mL) along with 1-iodopentane (1.5 mL) andmethanol (1 mL). The reaction was left for 1 week before solventextraction (×3) between chloroform (75 mL) and water (70 mL). Theorganic layer was dried with magnesium sulphate before being removedunder reduced pressure. The product was a white solid which was dried atroom temperature under vacuum.

Yield=0.077 g (77%)

Melting point: 107.9-108.3° C.

¹H NMR (Methanol-d₄) δ ppm: 0.67 (s 3H Me-18) 0.83-1.05 (m 10H Me-21plus CH₂) 1.0-2.439 (m steroid structure) 3.04 (s 6H CH₂) 3.47 (m 1H3CH) 3.76 (s 1H 7CH) 3.92 (d J=3.3 Hz 1H 12CH)

¹³C NMR (DMSO) δ ppm: 170.89 (C═O), 154.50, 71.01 (C12), 70.42 (C3),66.22 (C7), 60.58, 57.47, 57.41 (CH₃), 53.14, 52.55, 52.46, 48.55,46.07, 45.02, 43.30, 41.52, 41.33, 40.93, 35.30, 35.22, 34.85, 29.49,28.47, 28.35, 27.30, 26.17 steroid ring), 45.74 (CH₃), 40.50 (CH₂),30.37 (CH₂), 34.34 (CH₂), 31.17 (CH₂), 22.79 (CH₂), 22.57 (C19), 20.03,17.12 (C21), 14.51, 13.84 (CH₂), 12.29 (C18).

MS (+APCI) m/z=563.4767

IR (KBr) ν=3381 2929 2871 1645 (C═O) 1533 cm⁻¹

Synthesis of 3-cholanamidopropyl-hexyl-dimethyl-ammonium iodide (29)

N-[3-(dimethylamino)propyl] cholanamide (12) (0.5 g, 1 mmol) wasdissolved in chloroform (15 mL) along with 1-iodohexane (2.1 mL, 6.8mmol) and methanol (1 mL). The reaction was left for 5 days beforesolvent was removed under reduced vacuum. The product was purified bywashing with diethyl ether was collected by filtration and dried at roomtemperature under vacuum. The product was an orange solid.

Yield=0.34 g (68%)

Melting point: 101.7-102.3° C.

¹H NMR (Methanol-d₄) δ ppm: 0.68 (s, 3H, Me-18) 0.90 (d, J=5.6 Hz, 6H)1.01 (d, J=5.6 Hz, 3H, Me-21) 1.0-2.43 (m, steroid structure) 1.15 (td,J=7.0, 0.8 Hz) 3.04 (s, 6H, CH₂) 3.47 (m, 1H, 3CH) 3.77 (s, 1H, 7CH)3.92 (s, 1H, 12CH)

¹³C NMR (DMSO) δ ppm: 172.86 (C═O), 70.96 (C12), 70.37 (C3), 66.20 (C7),62.99, 60.98, 50.12, (45.98, 35.46, 35.13, 30.05, 29.48, 28.54, 27.26,26.20 steroid ring), 45.68 (CH₃), 34.34 (CH₂), 30.65 (CH₂), 31.55 (CH₂),25.40, 22.59 (C19), 21.87 (CH₂), 21.59, 17.10 (C21), 13.81 (CH₃), 12.32(C18), 9.10 (CH₂).

MS (+APCI) m/z=Found 577.4949; calculated for C₃₅H₆₅N₂O₄ 577.4939; 1.8ppm

IR (KBr) ν=3390, 2925, 2859, 1654 (C═O) cm⁻¹

Synthesis of 3-cholanamidopropyl-benzyl-dimethyl-ammonium iodide (30)

N-[3-(dimethylamino)propyl] cholanamide (12) (0.5 g, 1 mmol) wasdissolved in chloroform (20 mL) along with 2-phenylethyl bromide (1.36mL, 5.4 mmol) and methanol (1 mL). The reaction was left for 24 hoursbefore solvent was removed using a rotary evaporator. The resultingsolid was washed with diethyl ether (10 mL) and dried under vacuum atroom temperature. The product was a white solid.

Melting point: 103.8-105° C.

¹H NMR (CDCl₃) δ ppm: 0.58 (s, 2H, Me-18) 0.81 (s, 2H, Me-19) 0.92 (d,3H, Me-21) 1.0-2.43 (m, steroid structure) 2.49 (p, J=1.9 Hz, 14H)2.55-2.77 (m, 2H), 2.99 (s, 8H) 3.15-3.27 (m, 1H) 3.49 (t, J=6.6 Hz, 1H,3CH) 3.60 (s, 1H, 7CH) 3.77 (s, 1H, 12CH) 4.05 (dd, J=22.2, 3.3 Hz, 1H,3OH) 4.10 (d, 1H, 7OH) 4.32 (d, J=4.0 Hz, 1H, 120H),

¹³C NMR (DMSO) δ ppm: 172.89 (C═O), (140.23, 128.25, 126.21, 125.98aromatic ring), 70.96 (C12), 70.38 (C3), 50.13, (45.68, 35.47, 34.85,34.83, 34.35, 32.36, 31.65, 26.20 steroid ring), 23.54 (CH₂), 22.54(C19), 17.10 (C21), 12.32 (C18).

MS (+APCI) m/z=583.4473

IR (KBr) ν=3362, 3059, 3023, 2925, 2859, 1645 (C═O) cm⁻¹

Synthesis of 3-cholanamidopropyl-dimethyl-octadecyl-ammonium iodide (31)

N-[3-(dimethylamino)propyl] cholanamide (12) (0.5 g, 1 mmol) wasdissolved in chloroform (15 mL) along with 1-iodo-octadecane (2.4 g, 5mmol) and methanol (1 mL). The reaction was left for 72 hours beforesolvent was removed using a rotary evaporator. The resulting solid waswashed with diethyl ether (10 mL) before being collected by filtrationand dried under vacuum at room temperature. The product was a yellowsolid.

Yield=0.30 g (60%)

Melting point: 115.3-116.4° C.

¹H NMR (Methanol-d₄) δ ppm: 0.68 (s, 3H, Me-18) 0.84 (s, 3H, Me-19) 1.01(d, 3H, Me-21) 1.0-2.43 (m, steroid structure) 1.26 (s, 9H) 2.23 (s, 7H)3.05 (s) 3.15 (t) 3.27 (m) 3.44 (m, 1H, CH-3OH) 3.76 (s, 1H, CH-7OH)3.92 (s, 1H, CH-120H)

¹³C NMR (DMSO) δ ppm: 172.30 (C═O), 70.98 (C12), 66.21 (C7), (46.21,45.70, 34.85, 34.35, 31.80, 31.27, 30.34, 29.43, 28.77, 28.51, 27.26,26.34, 26.17 steroid ring), 22.56 (C19), 22.06, 17.04 (C21), 13.91(CH₂), 12.29 (C18).

MS (+APCI) m/z=754.6801

IR (KBr) ν=3362, 2922, 2850, 1639 (C═O) cm⁻¹

Synthesis of3-cholanamidopropyl-(2,2-dimethylpropanoyloxymethyl)-dimethyl-ammoniumchloride (32)

N-[3-(dimethylamino)propyl] cholanamide (12) (0.5 g, 1 mmol) wasdissolved in chloroform (20 mL) along with chloromethyl pivalate (1.44g, 5 mmol) and methanol (1 mL). The reaction was left for 12 hoursbefore solvent was removed using a rotary evaporator. The product waspurified by washing with diethyl ether before being collected byfiltraction and dried at room temperature under vacuum. The product wasa white solid.

Yield=0.335 g (66%)

Melting point: 127.9-128.3° C.

¹H NMR (CDCl₃) δ ppm: 0.57 (s, 3H, Me-18) 0.80 (s, 3H, Me-19) 1.08 (d,3H, Me-21) 1.0-2.43 (m, steroid structure) 2.70 (s) 3.05 (s) 3.36 (dd,J=15.7, 8.7 Hz, CH-3) 3.60 (s, 1H, CH-7) 4.01 (d, J=3.3 Hz, 1H, 3OH)4.10 (d, J=4.1 Hz, 1H, 7OH) 4.34 (d, J=4.1 Hz, 1H, 120H) 5.25 (s, 2H)5.85 (s, 1H) 7.93 (t, J=5.7 Hz, 1H, NH)

¹³C NMR (DMSO) δ 179.30 (C═O), 172.96 (C═O), 70.96 (C12), 66.20 (C7),54.42 (CH₃), 47.85, (46.00, 45.70, 41.92, 34.86, 34.35, 27.24, 26.98,26.43 steroid ring), 24.19, 22.59 (C19), 17.09 (C21), 12.31 (C18).

MS (+APCI) m/z=607.4669

IR (KBr) ν=3402, 3362, 2932, 2871, 2470, 1760, 1706, 1624 (C═O) cm⁻¹

Synthesis of 3-cholanamidopropyl-dimethyl-(2-phenylethyl)ammoniumbromide (33)

N-[3-(dimethylamino)propyl] cholanamide (12) (0.5 g, 1 mmol) wasdissolved in chloroform (20 mL) along with 1-bromo-3-phenylpropane (1.52mL, 5 mmol) and methanol (1 mL). The reaction was left for 12 hoursbefore solvent was removed using a rotary evaporator. The product waspurified by washing with diethyl ether (10 mL) was collected byfiltration and dried at room temperature under vacuum. The product was awhite solid.

Yield=0.4 g (80%)

Melting point: 176.8-177.4° C.

¹H NMR (DMSO) δ ppm: 0.58 (s, 3H, Me-18) 0.82 (s, 3H, Me-19) 0.95 (d,J=5.6 Hz, 3H, Me-21) 1.10 (d, J=7.0 Hz) 3.12 (d, J=14.5 Hz) 3.61 (m)3.78 (m) 4.00 (d, 1H, 3OH) 4.10 (d, 1H, 7OH) 4.34 (d, 1H, 120H) 7.28 (m,8H, aromatic) 7.93 (m, 1H, NH)

¹³C NMR (DMSO) δ: 172.24 (C═O), (133.08, 128.86, 127.54 aromatic ring),35.10, 34.35, 22.60 (C19), 17.12 (C21), 12.31 (18).

MS (+APCI) m/z=611.4411

IR (KBr) ν=3373, 3026, 2929, 2859, 1691 (C═O), 1645 cm⁻¹

Synthesis of 3-cholanamidopropyl-allyl-dimethyl-ammonium;bromide (34)

N-[3-(dimethylamino)propyl] cholanamide (12) (0.5 g, 1 mmol) wasdissolved in chloroform (20 mL) along with allyl bromide (1.52 mL, 5mmol) and methanol (1 mL). The reaction was left for 12 hours beforesolvent was removed using a rotary evaporator. The product was purifiedby washing with diethyl ether was collected by filtration and dried atroom temperature under vacuum. The product was a white solid.

Yield=0.19 g (40%)

Melting point: 121.3-121.9° C.

¹H NMR (CDCl₃) δ ppm: 0.68 (s, 3H, Me-18) 0.89 (s, 3H, Me-19) 0.85-1.61(m, 29H) 1.00 (d, 3H, Me-21) 1.0-2.43 (m, steroid structure) 3.04 3.76(s, 1H, CH-3) 3.87-4.13 (m, 6H) 5.61-5.76 (m, 3H) 5.93-6.17 (m, 2H),7.88 (s, 1H, NH)

¹³C NMR (DMSO) δ ppm: 172.88 (C═O), 127.60 (C═C), 125.76 (C═C), 70.96(C12), 70.37 (C3), 66.20 (C7), 65.11, 61.12, 49.73, (45.97, 45.68,35.26, 34.35, 28.53, 27.29, 26.20 steroid ring), 31.55 (CH₂—Br), 25.04(CH₂), 22.56 (C19), 17.11 (C21), 12.31 (C18).

MS (+APCI) m/z=Found 533.4307; calculated for C₃₂H₅₇N₂O₄ 533.4313; −1.1ppm

IR (KBr) ν=3368, 3077, 2925, 2862, 2710, 1733, 1651 (C═O), 1633, 1466cm⁻¹

Synthesis of 3-cholanamidopropyl-cyclopentyl-dimethyl-ammonium bromide(35)

N-[3-(dimethylamino)propyl] cholanamide (12) (0.5 g, 1 mmol) wasdissolved in DCM (10 mL) along with) of cyclopentyl bromide (0.9 mL, 10mmol). The reaction was left for 72 hours where the product preciptatedout. It was collected by filtration and dried at room temperature undervacuum. The product was a white solid.

Melting point: 102.6-103° C.

¹H NMR (DMSO) δ ppm: 0.57 (d, J=1.4 Hz, 3H Me-18) 0.76-0.97 (m, 6H,Me-19 and Me-21) 1.0-2.43 (m, steroid structure) 2.13 (s, dimethyl) 3.14(s, 3H) 3.4-3.55 (m, 1H, CH-3OH) 3.60 (s 1H CH-7OH) 3.77 (s, 1H,CH-120H) 3.94-4.18 (m, 4H) 4.32 (t, J=3.9 Hz, 1H) 5.35 (s, 1H, NH)

¹³C NMR (DMSO) δ ppm: 216.00, 138.38 (C═O), 70.92 (C19), 70.38 (C21),66.17 (C18), 60.48 (CH—Br), (45.71, 43.19, 41.39, 40.51, 40.17, 36.53,35.28, 34.89, 34.35, 30.64, 30.37, 30.35, 30.18, 28.51, 26.19 steroidring), 25.66, 24.51 (CH₂ ring), 22.59 (C19), 21.64, 16.91 (C21), 12.29(C18).

MS (+APCI) m/z=562.4448

IR (KBr) ν=3368, 2932, 2865, 2355, 1715 (C═O), 1578, 1460 cm⁻¹

Synthesis of 3-cholanamidoethylyl(trimethyl)ammonium iodide (36)

N-[3-(dimethylamino)ethyl] cholanamide (21) (0.5 g, 1.0 mmol) wasdissolved in DCM (20 mL) along with methyl iodide (0.3 mL, 2.1 mmol).The reaction was left for 12 hours where upon a solid formed and thesolvent had evaporated. Diethyl ether (10 mL) was added to the flask andthe solid was collected by filtration before being washed withchloroform (10 mL). The product was dried at room temperature undervacuum.

Yield=0.314 g (63%)

Melting point: 215.3-216.1° C.

¹H NMR (D₂O) δ ppm: 0.70 (s, 3H, Me-18) 0.90 (s, 3H, Me-19) 0.96 (d,J=Hz, 3H, Me-21) 1.0-2.43 (m, steroid structure) 3.11 (s, 9H, 3CH₃) 3.30(ddt, J=17.8 Hz) 3.48 (dd, J=10.4, 5.3 Hz, 1H, CH-3) 3.89 (s, 1H, CH-7)4.05 (s, 1H, CH-12) 7.65 (s, 1H, NH)

¹³C NMR (DMSO) δ ppm: 212.03, 172.84 (C═O), 79.97, 70.95 (C12), 70.37(C3), 66.20 (C7), 52.27 (CH₃), 52.18, 45.68 (CH₂), 40.52 (CH₂), (40.43,40.18, 40.10, 35.27, 34.35, 32.33, 29.95 steroid ring), 22.97, 22.60(19), 17.11 (C21), 12.31 (C18), 6.83.

MS (+APCI) m/z=Found 493.3991; calculated for C₂₉H₅₃N₂O₄ 493.4000; −1.8ppm

IR (KBr) ν=3423, 3241, 2935, 2862, 2252, 2118, 1663, 1618, 1539 cm⁻¹

Synthesis of 3-cholanamidoethylyl-ethyl-dimethyl-ammonium iodide (37)

N-[3-(dimethylamino)ethyl] cholanamide (21) (0.5 g, 1.0 mmol) wasdissolved in DCM (20 mL) along with ethyl iodide (0.4 mL, 1.3 mmol) andmethanol (1 mL). The reaction was left for 12 hours before the solventwas removed under reduced pressure. The solid was washed with ether (10mL) and chloroform (10 mL) before drying. The product was a yellowsolid.

Yield=0.280 g (56%)

Melting point: 142.3-142.7° C.

¹H NMR (D₂O) δ ppm: 0.72 (s, 3H, Me-18) 0.92 (s, 3H, Me-19) 0.97 (d, 3H,Me-21) 1.0-2.43 (m, steroid structure) 3.04 (s, 6H, CH₂) 3.21-3.34 (m,4H, 2CH₂) 3.38 (q, J=7.3 Hz, 2H, CH₂) 3.44-3.62 (m, 1H, CH-3) 3.89 (s,1H, CH-7) 4.06 (s, 1H, CH-12) 7.67 (s, 1H, NH)

¹³C NMR (DMSO) δ ppm: 197.83, 172.88 (C═O), 70.96 (C12), 70.37 (C3),66.20 (C7), 60.46, 58.59 (CH₃), 49.59, 49.52, (45.96, 41.46, 41.38,40.51, 40.17, 39.84, 39.51, 39.42, 39.17, 39.01, 38.84, 38.51, 35.47,35.25, 35.15, 34.87, 34.34, 32.35, 30.35, 28.55, 27.26, 26.20 steroidring), 45.68 (CH₂), 31.52 (CH₂), 22.77, 22.59 (C19), 22.52 (CH₂), 17.10(C21), 12.31 (C18), 7.76.

MS (+APCI) m/z=Found 507.4147; calculated for C₃₀H₅₅N₂O₄ 507.4156; −1.8ppm

IR (KBr) ν=3387, 2932, 2862, 2689, 1706, 1648 (C═O), 1533 cm⁻¹

Synthesis of 3-cholanamidopropyl-propyl-dimethyl-ammonium iodide (38)

N-[3-(dimethylamino)ethyl] cholanamide (21) (0.5 g, 1.0 mmol) wasdissolved in DCM (20 mL) along with 1-iodopropane (0.5 mL, 1.7 mmol) andmethanol (1 mL). The reaction was left for 6 days before the solvent wasremoved under reduced pressure. The solid was washed with diethyl ether(10 mL) and collected by filtration. Further purification was carriedout by washing the crude product with diethyl ether (2×10 mL) andchloroform (2×10 mL). The product was a white solid which was dried atroom temperature under vacuum.

Yield=0.283 g (56%)

Melting point: 108.1-108.9° C.

¹H NMR (DMSO) δ ppm: 0.57 (s, 3H, Me-18) 0.79 (s, 3H, Me-19) 0.86-0.94(m, 37H, Me-21 plus 2CH₂) 1.0-2.43 (m, steroid structure) 2.94-3.28 (m,14H) 3.61 (s, 1H, 7CH) 3.77 (s, 1H, 12CH) 3.99 (d, J=, 1H 3OH) 4.10 (d,J=, 1H, 7OH) 4.32 (d, J=4.0 Hz, 1H, 120H) 7.88 (t, J=5.7 Hz, NH)

¹³C NMR (DMSO) δ ppm: 172.92 (C═O), 70.97 (C12), 70.37 (C3), 66.20 (C7),64.37, 61.10, 50.20, 50.16, (45.97, 35.45, 35.15, 34.86, 34.34, 32.37,30.34, 28.53, 26.20 steroid ring), 45.68 (CH₃), 31.54 (CH₂), 27.26(CH₂), 22.58 (C19), 17.10 (C21), 15.32 (CH₃), 12.31 (C18), 10.45 (CH₂).

MS (+APCI) m/z=Found 521.4303; calculated for C₃₁H₅₇N₂O₄ 521.4313; −1.9ppm

IR (KBr) ν=3390, 2935, 2862, 2243, 2121, 1651 (C═O), 1536 cm⁻¹

Synthesis of 3-cholanamidoethyl-butyl-dimethyl-ammonium iodide (39)

N-[3-(dimethylamino)propyl] cholanamide (21) (0.5 g, 1.0 mmol) wasdissolved in DCM (20 mL) along with 1-iodobutane (0.6 mL, 2 mmol) andmethanol (1 mL). The reaction was left for 3 days where a precipitateformed. The crude product was collected by filtration and washed withchloroform (15 mL). The product was a white solid which was dried atroom temperature under vacuum.

Yield=0.338 g (67%)

Melting point: 186.4-187.4° C.

¹H NMR (D₂O) δ ppm: 0.68 (s, 3H, Me-18) 0.88 (s, H, Me-19) 0.84-1.00 (m,5H, Me-21 plus CH₂) 1.0-2.43 (m, steroid structure) 3.02 (s, 6H, CH₂)3.14-3.32 (m, 6H, CH₂) 3.47 (m, 1H, 3CH) 3.86 (s, 1H, 7CH) 4.02 (s, 1H,12CH)

MS (+APCI) m/z=Found 535.4458; calculated for C₃₂H₅₉N₂O₄ 535.4469; −2.1ppm

IR (KBr) ν=3484, 3432, 3250, 3071, 2922, 2865, 1633 (C═O), 1551 cm⁻¹

Synthesis of 3-cholanamidoethyl-pentyl-dimethyl-ammonium iodide (40)

N-[3-(dimethylamino)ethyl] cholanamide (21) (0.5 g, 1 mmol) wasdissolved in DCM (20 mL) along with 1-iodopentane (0.6 mL, 1.9 mmol) andmethanol (1 mL). The reaction was left for 3 days where a precipitateformed. The product was collected by filtration, washed with chloroform(20 mL) and dried at room temperature under vacuum. The product was awhite solid.

Yield=0.396 g (79%)

Melting point: 139.6-141.2° C.

¹H NMR (Methanol-d₄) δ ppm: 0.67 (s 3H Me-18) 0.83-1.05 (m 10H Me-21plus CH₂) 1.0-2.439 (m steroid structure) 3.04 (s 6H CH₂) 3.47 (m 1H3CH) 3.76 (s 1H 7CH) 3.92 (d J=3.3 Hz 1H 12CH)

¹³C NMR (DMSO) δ ppm: 170.89 (C═O), 154.50, 71.01 (C12), 70.42 (C3),66.22 (C7), 60.58, 57.47, 57.41 (CH₃), 53.14, 52.55, 52.46, 48.55,46.07, 45.02, 43.30, 41.52, 41.33, 40.93, 35.30, 35.22, 34.85, 29.49,28.47, 28.35, 27.30, 26.17 steroid ring), 45.74 (CH₃), 40.50 (CH₂),30.37 (CH₂), 34.34 (CH₂), 31.17 (CH₂), 22.79 (CH₂), 22.57 (C19), 20.03,17.12 (C21), 14.51, 13.84 (CH₂), 12.29 (C18).

MS (+APCI) m/z=Found 549.4618; calculated for C₃₃H₆₁N₂O 4549.4626; −1.4ppm

IR (KBr) ν=3484, 3250, 3071, 2925, 2868, 1627 (C═O), 1560 cm⁻¹

Synthesis of 3-cholanamidoethyl-hexyl-dimethyl-ammonium iodide (41)

N-[3-(dimethylamino)ethyl] cholanamide (21) (0.5 g, 1 mmol) wasdissolved in DCM (10 mL) along with 1-iodohexane (0.7 mL, 2 mmol) andmethanol (1 mL). The reaction was left for 3 days where a precipitateformed. The product was collected by filtration, washed with chloroform(20 mL) and dried at room temperature under vacuum. The product was awhite solid.

Yield=0.45 g (91%)

Melting point: 104.2-104.5° C.

¹H NMR (Methanol-d₄) δ ppm: 0.68 (s, 3H, Me-18) 0.90 (d, J=5.6 Hz, 6H)1.01 (d, J=5.6 Hz, 3H, Me-21) 1.0-2.43 (m, steroid structure) 1.15 (td,J=7.0, 0.8 Hz) 3.04 (s, 6H, CH₂) 3.47 (m, 1H, 3CH) 3.77 (s, 1H, 7CH)3.92 (s, 1H, 12CH)

¹³C NMR (DMSO) δ ppm: 172.86 (C═O), 70.96 (C12), 70.37 (C3), 66.20 (C7),62.99, 60.98, 50.12, (45.98, 35.46, 35.13, 30.05, 29.48, 28.54, 27.26,26.20 steroid ring), 45.68 (CH₃), 34.34 (CH₂), 30.65 (CH₂), 31.55 (CH₂),25.40, 22.59 (C19), 21.87 (CH₂), 21.59, 17.10 (C21), 13.81 (CH₃), 12.32(C18), 9.10 (CH₂).

MS (+APCI) m/z=Found 563.4771; calculated for C₃₄H₆₃N₂O₄ 563.4782; −2.0ppm

IR (KBr) ν=3393, 3238, 3056, 2925, 2853, 1703, 1651 (C═O), 1606 cm⁻¹

Synthesis of 3-cholanamidoethyl-allyl-dimethyl-ammonium bromide (42)

N-[3-(dimethylamino)ethyl] cholanamide (21) (0.5 g, 1 mmol) wasdissolved in DCM (10 mL) along with allyl bromide (0.4 mL, 3.3 mmol) andmethanol (1 mL). The reaction was left for 12 hours before solvent wasremoved using a rotary evaporator. The product was purified by washingwith diethyl ether was collected by filtration and dried at roomtemperature under vacuum. The product was a white solid.

Yield=0.129 g (26%)

Melting point: 119.1-120.3° C.

¹H NMR (CDCl₃) δ ppm: 0.68 (s, 3H, Me-18) 0.89 (s, 3H, Me-19) 0.85-1.61(m, 29H) 1.00 (d, 3H, Me-21) 1.0-2.43 (m, steroid structure) 3.04 3.76(s, 1H, CH-3) 3.87-4.13 (m, 6H) 5.61-5.76 (m, 3H) 5.93-6.17 (m, 2H),7.88 (s, 1H, NH)

¹³C NMR (DMSO) δ ppm: 172.88 (C═O), 127.60 (C═C), 125.76 (C═C), 70.96(C12), 70.37 (C3), 66.20 (C7), 65.11, 61.12, 49.73, (45.97, 45.68,35.26, 34.35, 28.53, 27.29, 26.20 steroid ring), 31.55 (CH₂—Br), 25.04(CH₂), 22.56 (C19), 17.11 (C21), 12.31 (C18).

MS (+APCI) m/z=Found 519.4146; calculated for C₃₁H₅N₂O₄ 519.4156; −2.0ppm

IR (KBr) ν=3362, 2929, 2862, 1700, 1645 (C═O), 1536 cm⁻¹

Synthesis of N-[2-(1-methylpyrrolidin-1-ium-1-yl)ethyl]cholanamideiodide (43)

N-(2-pyrrolidin-1-ylethyl)cholanamide (10) (0.5 g, 1 mmol) was dissolvedin DCM (10 mL) along with of methyl iodide (1.4 mL, 10 mmol). Thereaction was left for 60 hours where the product precipitated out. Itwas then collected by filtration, washed with chloroform (20 mL) anddried at room temperature under vacuum. The product was a white solid.

Melting point: 140.7-141-4° C.

¹H NMR (DMSO) δ ppm: 0.58 (s, 3H, Me-18) 0.81 (s, 3H, Me-19) 0.93 (d,J=5.8 Hz, 3H, Me-21) 1.0-2.43 (m, steroid structure) 2.23 (s) 2.55 2.643.44 (1H, CH-3OH) 3.84 (s, 1H, CH-7OH) 3.96 (s, 1H, CH-120H) 6.40 (s,1H, NH)

¹³C NMR (DMSO) δ: 173.30 (C═O), 70.96 (C12), 70.37 (C3), 66.19 (C7),63.86 (CH₂), 61.67 (CH₂ ring), 47.44, (45.98, 45.68, 41.46, 35.09,34.34, 33.58, 32.25, 31.40, 28.54, 27.26, 26.20 steroid ring), 41.39(CH₂), 22.76 (CH₂ ring), 22.59 (C19), 20.91, 17.04 (C21), 12.31 (C18).

MS (+APCI) m/z=Found 519.4152; calculated for C₃₁H₅₅N₂O 4519.4156; −0.8ppm

IR (KBr) ν=3390, 3274, 2922, 2862, 1651 (C═O), 1533, 1460 cm⁻¹

Synthesis of N-[2-(1-ethylpyrrolidin-1-ium-1-yl)ethyl]cholanamide iodide(44)

N-(2-pyrrolidin-1-ylethyl)cholanamide (10) (0.5 g, 1 mmol) was dissolvedin DCM (10 mL) along with of ethyl iodide (0.4 mL, 10 mmol). Thereaction was left for 60 hours where the product precipitated out. Itwas collected by filtration, washed with chloroform and dried at roomtemperature under vacuum. The product was a yellow solid.

Melting point: 120.7-121.7° C.

¹H NMR (DMSO) δ ppm: 0.57 (s, 3H, Me-18) 0.80 (s, 3H, Me-19) 0.92 (d,J=6.2 Hz, 3H, Me-21) 1.0-2.43 (m, steroid structure) 2.23 (d, J=12.1 Hz)2.55 2.64 3.44 (1H, CH-3OH) 3.60 (s, 1H, CH-7OH) 3.77 (s, 1H, CH-120H)5.75 8.08 (s, 1H, NH)

Carbon N/A

MS (+APCI) m/z=Found 533.4302; calculated for C₃₂H₅₇N₂O₄ 533.4313; −2.0ppm

IR (KBr) ν=3378, 2929, 2862, 2361, 2158, 2018, 1645 (C═O), 1527 cm⁻¹

Synthesis ofN-[2-(1-propylpyrrolidin-1-ium-1-yl)ethyl]cholanamide;iodide (45)

N-(2-pyrrolidin-1-ylethyl)cholanamide (10) (0.5 g, 1 mmol) was dissolvedin DCM (15 mL) along with of 1-iodopropane (3 mL, 10 mmol). The reactionwas left for 12 hours where the product preciptated out. It wascollected by filtration, washed with chloroform and dried. The productwas a yellow solid.

Yield=0.947 g

Melting point: 203.8-204.1° C.

¹H NMR (D₂O) δ ppm: 0.68 (s, 3H, Me-18) 0.80 (s, 3H, Me-19) 0.91 (d, 3H,Me-21) 1.0-2.43 (m, steroid structure) 3.18 (m) 3.49 (ddt, J=40.0, 13.4,6.6 Hz, 8H, CH-3OH, CH₂) 3.87 (s, 1H, CH-7OH) 4.03 (s, 1H, CH-120H) 7.64(s, 1H, NH)

Carbon=N/A

MS (+APCI) m/z=Found 547.4463; calculated for C₃₃H₅₉N₂O₄ 547.4469; −1.2ppm

IR (KBr) ν=3566, 3353, 3217, 2913, 2856, 2246, 2121, 1642, 1527 cm⁻¹

Synthesis of N-[2-(1-hexylylpyrrolidin-1-ium-1-yl)ethyl]cholanamideiodide (48)

N-(2-pyrrolidin-1-ylethyl)cholanamide (10) (0.5 g, 1 mmol) was dissolvedin DCM (15 mL) along with of 1-iodopentane (2 mL, 10 mmol). The reactionwas left for 96 hours where the product precipitated out. It wascollected by filtration, washed with chloroform (15 mL) and dried atroom temperature under vacuum. The product was a yellow solid.

Yield=0.49 g (98%)

¹H NMR (CDCl₃) δ ppm: 0.58 (s, 3H, Me-18) 0.81 (s, 3H, Me-19) 0.91 (d,3H, Me-21) 1.0-2.43 (m, steroid structure) 2.50 (p, J=1.8 HZ, 4H) 3.33(m) 3.49 (m, 8H, CH-3OH, CH₂) 3.77 (s, 1H, CH-7OH) 4.32 (s, 1H, CH-120H)7.72 (s, 1H, NH), 8.08, 8.31

¹³C NMR (DMSO) δ 173.38 (C═O), 70.96 (C12), 70.37 (C3), 70.30, 66.19(C7), 62.48, 58.76, 56.74 (CH₂ ring), (45.95, 45.69, 40.48, 40.15,39.81, 39.48, 39.15, 38.81, 38.48, 35.09, 34.34, 32.22, 31.36, 30.71,28.54, 27.25, 26.21 steroid ring), 25.43 (CH₂ ring), 22.59 (C19), 21.91(CH₂), 21.15, 17.04 (C21), 13.82 (CH₃), 12.29 (C18).

MS (+APCI) m/z=589.4926

IR (KBr) ν=3466, 3362, 3186, 3044, 2953, 2922, 1706, 1660, 1624 (C═O),1530 cm⁻¹

Synthesis of N-[2-(1-allylpyrrolidin-1-ium-1-yl)ethyl]cholanamidebromide (49)

N-(2-pyrrolidin-1-ylethyl)cholanamide (10) (0.5 g, 1 mmol) was dissolvedin chloroform (20 mL) along with allylbromide (1.2 mL, 10 mmol). Thereaction was left for 96 hours where the product precipitated out. Itwas collected by filtration, washed with chloroform (20 mL) and dried atroom temperature under vacuum. The product was a white solid.

Yield=0.59 g (60%)

Melting point: 186.5-186.9° C.

¹H NMR (DMSO) δ ppm: 0.59 (s, 3H, Me-18) 0.88 (s, 3H, Me-19) 0.94 (d,3H, Me-21) 1.0-2.43 (m, steroid structure) 2.07 (m) 2.23 (d, J=12.1 Hz,CH₂) 2.55 2.64 3.52 (m, 3H, CH-3OH+CH₂) 3.61 (s, 1H, CH-7OH) 3.79 (s,1H, CH-120H) 4.00 (d, 2H, CH₂ allyl bromide) 5.66 (t, 2H, allyl bromide)6.08 (m, 1H, allyl bromide) 8.13 (t, 1H, NH) 8.32 (s, 1H)

¹³C NMR (DMSO) δ ppm: 173.37 (C═O), 127.23 (C═C), 126.28 (C═C), 79.17,70.94 (C12), 70.36 (C3), 66.18 (C3), 61.74, 60.54, 57.71 (CH₂), (45.94,45.68, 40.48, 40.15, 40.07, 39.82, 39.73, 39.48, 39.15, 38.81, 38.48,34.34, 33.11, 31.39, 30.35, 28.54, 28.53, 27.29, 26.20 steroid ring),32.18 (CH₂), 22.60 (C19), 21.16, 17.03 (C21), 12.30 (C18).

MS (+APCI) m/z=Found 545.4306; calculated for C₃₃H₅₇N₂O₄ 545.4313; −1.3ppm

IR (KBr) ν=3472, 3365, 3211, 3050, 2922, 2859, 2458, 2042, 1630 (C═O),1542 cm⁻¹

Synthesis of N-[2-(1-benzylpyrrolidin-1-ium-1-yl)ethyl]cholanamidebromide (50)

N-(2-pyrrolidin-1-ylethyl)cholanamide (10) (0.5 g, 1 mmol) was dissolvedin DCM (15 mL0 along with benzyl bromide (1.2 mL, 10 mmol). The reactionwas left for 48 hours where the product precipitated out. It wascollected by filtration, washed with chloroform (15 mL) and dried atroom temperature under vacuum. The product was a yellow solid.

Yield=0.301 g (30%)

Melting point: 165.7-165.9° C.

¹H NMR (D₂O) δ ppm: 0.56 (s, 3H, Me-18) 0.87 (s, 3H, Me-19) 0.92 (d, 3H,Me-21) 1.0-2.43 (m, steroid structure) 2.19 (s) 2.23 (d, J=12.1 Hz, CH₂)3.37-3.68 (m, 6H, CH-3OH+CH₂) 3.75 (s, 1H, CH-7OH) 3.98 (s, 1H, CH-120H)7.38 (s, 1H, NH) 7.54 (m, aromatic)

¹³C NMR (DMSO) δ ppm: 173.43 (C═O), (132.61, 129.04, 128.41 aromaticring), 60.89 (CH₂), 60.86 (CH₂), (45.69, 35.25, 35.10, 34.94, 34.67,34.34 steroid ring), 22.59 (C19), 20.86, 17.03 (C21), 12.28 (C18).

MS (+APCI) m/z=595.4461

IR (KBr) ν=3368, 3053, 2922, 2862, 2361, 1788, 1706, 1648 (C═O), 1536cm⁻¹

Synthesis ofN-[2-[1-(3-phenylpropyl)pyrrolidin-1-ium-1-yl]ethyl]cholanamide bromide(51)

N-(2-pyrrolidin-1-ylethyl)cholanamide (10) (0.5 g, 1 mmol) was dissolvedin DCM (15 mL) along with 1-bromo-3-phenylpropane (2 mL, 10 mmol). Thereaction was left for 12 hours where the product precipitated out. Itwas collected by filtration, washed with chloroform (10 mL) and dried atroom temperature under vacuum. The product was a yellow solid.

Yield=0.986 g (98%)

Melting point: 140.8-141.3° C.

¹H NMR (DMSO) δ ppm: 0.57 (s, 3H, Me-18) 0.77 (s, 3H, Me-19) 0.92 (d,J=5.9 Hz, 3H, Me-21) 1.0-2.43 (m, steroid structure) 2.68, (dt, J=10.9,7.4 Hz, 2H, CH₂) 3.16-3.58 (m, 9H, CH-3OH, CH₂) 3.60 (s, 1H, CH-7OH)3.77 (s, 1H, CH-120H) 7.12-7.36 (m, 4H, aromatic) 8.11 (t, J=5.5 Hz, 1H,NH)

¹³C NMR (DMSO) δ ppm: 173.32 (C═O), (140.52, 128.37, 128.37, 128.33,125.98 aromatic ring), 79.15, 70.97 (C12), 70.38 (C3), 66.20 (C7), 63.84(CH₂), 61.68 (CH₂), 47.42, (45.98, 45.69, 45.68, 40.48, 40.14, 39.81,39.48, 39.14, 38.81, 38.47, 35.10, 34.86, 34.35, 33.85, 33.58, 33.36,32.25, 31.39, 30.34, 28.53, 27.27, 26.20 steroid ring), 34.34 (CH₂),22.59 (C19), 20.90, 17.04 (C21), 12.30 (C18).

MS (+APCI) m/z=Found 519.4150; expected 623.478 (MI− CH₂CH₂C₆H₆)

IR (KBr) ν=3387, 3256, 3068, 3026, 2929, 2868, 1651, 1624 (C═O), 1533cm⁻¹

Synthesis of [1-(2-cholanamidoethyl)pyrrolidin-1-ium-1-yl]methyl2,2-dimethylpropanoate chloride (52)

N-(2-pyrrolidin-1-ylethyl)cholanamide (10) (0.5 g, 1 mmol) was dissolvedin DCM (10 mL) along with chloromethyl pivalate (1.4 mL, 10 mmol). Thereaction was left for 72 hours where the product precipitated out. Itwas collected by filtration, washed with chloroform and (15 mL) dried atroom temperature under vacuum. The product was a yellow solid.

Yield=0.335 g (34%)

Melting point: 121.2-121.8° C.

¹H NMR (DMSO) δ ppm: 0.68 (s 3H Me-18) 0.80 (s 3H Me-19) 0.91 (d 3HMe-21) 1.0-2.439 (m steroid structure) 3.38 (t J=9.1 Hz 1H CH-3) 3.87 (s1H CH-7OH) 4.03 (s 1H CH-120H) 8.66 (s 1H NH) 11.39 (s 1H)

¹³C NMR (DMSO) δ 206.43, 179.27, 175.20 (C═O), 173.36, 173.14, 77.70,70.93 (C12), 70.37 (C3), 69.58, 66.19 (C7), 60.88, 57.80, 53.07 (CH₂),52.88 (CH₂ ring), (45.71, 37.66, 35.16, 34.85, 34.34, 33.32, 33.29,32.16, 31.29, 30.65, 30.65, 28.50, 26.97, 26.97, 26.88, 26.42, 26.31,26.20 steroid ring), 22.57 (C19), 21.81 (CH₂ ring), 17.07 (C21), 12.28(C18).

MS (+APCI) m/z=619.4647

IR (KBr) ν=3368, 2929, 2874, 2607, 2476, 2249, 2118, 1754, 1709, 1651(C═O), 1539 cm⁻¹

Synthesis of N-(1-methyl-1-phenyl-piperidin-1-ium-4-yl)cholanamideiodide (53)

N-(1-phenyl-4-piperidyl) cholanamide (13) (0.5 g, 1 mmol) was dissolvedin DCM (15 mL) along with methyl iodide (1.4 mL, 10 mmol). The reactionwas left for 72 hours where the product precipitated out. It wascollected by filtration, washed with DCM (20 mL) and dried at roomtemperature under vacuum. The product was a yellow solid.

Yield=0.7 g (70%)

Melting point: 165.8-166.7° C.

¹H NMR (D₂O) δ ppm: 0.58 (s, 3H, Me-18) 0.79 (s, 3H, Me-19) 0.92 (d, 3H,Me-21) 1.0-2.43 (m, steroid structure) 2.82-3.18 (m, 7H) 3.43 (m, 1H,CH-3) 3.60 (s, 1H, CH-7) 3.78 (s, 1H, CH-12) 4.38-4.54 (m, 2H, CH₂) 4.60(m, 2H, CH₂) 7.53 (m, 5H, aromatic) 7.79-7.90 (m, 1H, NH)

¹³C NMR (DMSO) δ ppm: 172.24 (C═O), (133.08, 128.86, 127.54 aromaticring), 35.10, 34.35, 22.60 (C19), 17.12 (C21), 12.31 (C18).

MS (+APCI) m/z=Found 595.4464; calculated for C₃₆H₅₇N₂O₄ 595.4469; 0.84ppm

IR (KBr) ν=3396, 2925, 2862, 2355, 1648 (C═O), 1533 cm⁻¹

Synthesis of N-(1-propyl-1-phenyl-piperidin-1-ium-4-yl)cholanamideiodide (54)

N-(1-phenyl-4-piperidyl) cholanamide (13) (0.5 g, 1 mmol) was dissolvedin DCM (15 mL) along with methyl iodide (1.5 mL, 10 mmol). The reactionwas left for 72 hours where the product precipitated out. It wascollected by filtration, washed with DCM (15 mL) and dried at roomtemperature under vacuum. The product was a yellow solid.

Yield=0.7 g (70%)

Melting point: 113-113.8° C.

¹H NMR (DMSO) δ ppm: 0.58 (d J=4.1 Hz 3H Me-18) 0.80 (s 3H Me-19) 0.91(m 9H Me-21+2CH₃) 1.0-2.439 (m steroid structure) 2.08 (s CH₂) 3.01-3.33(m 9H CH₂) 3.61 (s 1H CH-7OH) 3.78 (s 1H CH-12OH) 3.92-4.11 (m 2H) 4.60(d J=10.3 Hz 1H) 7.42-7.62 (m 6H aromatic ring)

Carbon=N/A

MS (+APCI) m/z=Found 651.5086; expected 609.4625 (MI+ 52)

IR (KBr) ν=3393, 2932, 2862, 2361, 2155, 2018, 1642 (C═O), 1533 cm⁻¹

Synthesis of N-(1-hexyl-1-phenyl-piperidin-1-ium-4-yl)cholanamide iodide(57)

N-(1-phenyl-4-piperidyl) cholanamide (13)(0.5 g, 1 mmol) was dissolvedin DCM (15 mL) along with 1-iodohexane (1.5 mL, 10 mmol). The reactionwas left for 120 hours where the product did not precipitate. Thesolvent was removed under vacuum and the product was sonicated thenwashed with diethyl ether (10 mL). The product was dried at roomtemperature under vacuum and it a yellow solid.

Yield=0.55 g (55%)

Melting point: 126.4-127.8° C.

¹H NMR (CDCl₃) δ ppm: 0.66 (s, 3H, Me-18) 0.89 (s, 3H, Me-19) 0.79 (d,Me-21) 1.0-2.43 (m, steroid structure) 2.04 (s) 2.83 (m) 3.47 (m, 1H,CH-3) 3.56 (m, CH₂) 3.81 (s, 1H, CH-7) 3.94 (s, 1H, CH-12) 6.06 (d,J=7.8 Hz, 1H, NH) 7.32 (q, J=4.3, 3.7 Hz, 6H, aromatic)

¹³C NMR (DMSO) δ ppm: 206.44, 171.82 (C═O), (128.73, 128.16, 126.95aromatic ring), 70.97 (C12), 70.46 (C3), 66.20 (C7), 51.90 (CH₂),(46.11, 45.69, 40.49, 40.16, 35.30, 34.86, 34.35, 34.35, 32.56, 31.72,30.66, 29.68, 28.53, 27.30, 26.18 steroid ring), 22.59 (C19), 17.11(C21), 12.31 (C18).

MS (+APCI) m/z=Found 665.5257; calculated for C₄₁H₆₇N₂O₄ 665.5252; 0.77ppm

IR (KBr) ν=3372, 3296, 3056, 3032, 2932, 2862, 2364, 1642 (C═O), 1530cm⁻¹

Synthesis of N-(1-benzyl-1-phenyl-piperidin-1-ium-4-yl)cholanamide (58)

N-(1-phenyl-4-piperidyl) cholanamide (13) (0.5 g, 1 mmol) was dissolvedin DCM (15 mL) along with benzyl bromide (1.5 mL, 10 mmol). The reactionwas left for 12 days where the product precipitated. The product washedwith diethyl ether (10 mL). The product was dried at room temperatureunder vacuum and it a peach solid.

Yield=0.1839 g (18%)

Melting point: 150.3-150.9° C.

¹H NMR (MeOD) δ ppm: 0.68 (s, 3H, Me-18) 0.90 (s, 3H, Me-19) 0.98 (d,3H, Me-21) 1.0-2.43 (m, steroid structure) 3.68 (m, 1H, CH-3) 3.78 (s,1H, CH-7) 3.92 (s, 1H, CH-12) 4.49 (s, 2H, CH₂) 7.19-7.67 (m, 11H,aromatics)

¹³C NMR (DMSO) δ ppm: (133.56, 133.08, 130.33, 129.23, 127.46, 127.12,126.37 aromatic ring), 70.96 (C12), 45.69, 34.35 (CH₂), 27.31, 24.50,22.59 (C19), 17.04 (C21), 12.30 (C18), 5.85, 1.58.

MS (+APCI) m/z=Found 671.4770; expected 657.4625 (MI+CH₃)

IR (KBr) ν=3411, 3329, 3062, 2929, 2859, 2671, 2355, 1654 (C═O), 1536,1493 cm⁻¹

Synthesis of (4-cholanamido-1-phenyl-piperidin-1-ium-1-yl)methyl2,2-dimethylpropanoate chloride (59)

N-(1-phenyl-4-piperidyl) cholanamide (13) (0.5 g, 1 mmol) was dissolvedin chloroform (15 mL) along with of chloromethyl pivalate (1.4 mL, 10mmol). The reaction was left for 12 days where the product did notprecipitate. The solvent was taken off under vacuum and the productsonicated and washed with diethyl ether (10 mL). The product was driedat room temperature under vacuum and it a yellow solid.

Yield=0.5215 g (52%)

Melting point: 135.9-137° C.

¹H NMR (MeOD) δ ppm: 0.70 (s, 3H, Me-18) 0.92 (s, 3H, Me-19) 1.02 (d,3H, Me-21) 1.0-2.439 (m, steroid structure) 2.99 (d, J=11.4 Hz, 1H) 3.78(s, 1H, CH-7) 3.97 (s, 1H, CH-12) 5.75 (s, 2H, O—CH₂—Cl) 7.24-7.45 (m,2H, aromatic) 7.55 (m, 1H, NH)

¹³C NMR (DMSO) δ ppm: 206.44 (C═O), 175.60 (C═O), 128.69, 69.59 (CH₂),)45.69, 35.12, 34.35, 30.66, 26.97, 26.32 steroid ring), 22.59 (C19),17.11 (C21), 12.30 (C18).

MS (+APCI) m/z=Found 695.4994; calculated for C₄₁H₆₅N₂O 6695.4993; 0.143ppm

IR (KBr) ν=3365, 3065, 2935, 2865, 2631, 2525, 2361, 1751, 1642 (C═O),1542 cm⁻¹

Synthesis ofN-[1-phenyl-1-(3-phenylpropyl)piperidin-1-ium-4-yl]cholanamide bromide(60)

N-(1-phenyl-4-piperidyl) cholanamide (13) (0.5 g, 1 mmol) was dissolvedin chloroform (15 mL) along with 1-bromo-3-phenylpropane (1.3 mL, 10mmol). The reaction was left for 12 days where the product did notprecipitate. The solvent was taken off under vacuum and the productsonicated and washed with diethyl ether (10 mL). The product was driedat room temperature under vacuum and it a yellow solid.

Yield=0.79 g (80%)

Melting point: 126.1-126.7° C.

¹H NMR (MeOD) δ ppm: 0.69 (s, 3H, Me-18) 0.91 (s, 3H, Me-19) 0.98 (d,3H, Me-21) 1.0-2.43 (m, steroid structure) 3.43 (m) 3.66 (m, 1H, CH-3)3.79 (s, 1H, CH-7) 3.94 (s, 1H, CH-12) 7.09-7.41 (m, 7H, aromatic)

¹³C NMR (DMSO) δ ppm: (140.52, 128.85, 128.43, 128.37, 128.33, 125.98aromatic ring), 70.99 C12), 70.38 (C3), 66.20 (C7), (45.95, 45.69,45.69, 40.49, 40.15, 35.27, 34.89, 33.36, 31.62, 30.38, 28.52, 27.31,26.19 steroid ring), 34.61 (CH₂), 34.36 (CH₂), 33.86 (CH₂), 22.59 (C19),17.12 (C21), 12.31 (C18).

MS (+APCI) m/z=Found 699.5087; calculated for C₄₄H₆₅N₂O₄ 695.5095; 1.143ppm

IR (KBr) ν=3375, 3023, 2932, 2859, 1642 (C═O), 1536 cm⁻¹

Synthesis of N-(1-allyl-1-phenyl-piperidin-1-ium-4-yl)cholanamidebromide (61)

N-(1-phenyl-4-piperidyl) cholanamide (13) (0.5 g, 1 mmol) was dissolvedin DCM (15 mL) along with allyl bromide (1.2 mL, 10 mmol). The reactionwas left for 4 days where the product precipitated then washed with DCM(25 mL). The product was dried at room temperature under vacuum and togive an orange solid.

Yield=0.242 g (24%)

Melting point: 161.4-161.7° C.

¹H NMR (CDCl₃) δ ppm: 0.58 (d, J=3.6 Hz, 2H, Me-18) 0.81 (s, 2H, Me-19)0.93 (d, J=5.8 Hz 3H, Me-21) 1.0-2.43 (m steroid structure) 3.18, 3.61(s, 1H, CH-3), 3.78 (s 1H CH-7), 3.95 (dd, J=16.7, 9.8 Hz 1H CH-12) 4.08(s) 4.31 (s 2HCH₂) 4.55-4.68 (m C═CH) 5.61-5.83 (m C═CH) 7.42-7.62 (m 6Haromatic ring)

¹³C NMR (DMSO) δ ppm: 187.15 (C═O), 133.09 (C═C), (131.49, 130.30,128.97, 127.16 aromatic ring), 92.56 (C═C), 70.96 (C12), 70.37 (C3),66.19 (C7), 55.64 (CH₂), (46.01, 41.48, 40.50, 40.16, 39.83, 39.49,39.15, 38.82, 38.74, 38.50, 37.34, 35.27, 30.66, 28.56 steroid ring),22.59 (C19), 17.13 (C21), 12.30 (C18).

MS (+APCI) m/z=Found 621.4620; calculated for C₃₈H₅₉N₂O₄ 621.4626; 0.96ppm

IR (KBr) ν=3378, 3056, 2935, 2856, 2552, 2358, 1639 (C═O), 1533 cm⁻¹

Synthesis of 3α, 7, 12α-triacrylate cholic acid methyl ester (62)

Methyl cholate (4.22 g, 10 mmol) was dissolved in dry chloroform (30mL). Triethylamine (2.23 mL, 16 mmol) was added and the flask was cooledto ice temperature. Acryloyl chloride (1.3 mL, 15 mmol) in chloroform(10 mL) was dripped in over 30 minutes. The mixture was protected fromthe light and left at room temperature overnight. More acryloyl chloride(1.3 mL, 15 mmol) in dry chloroform (10 mL) was dripped in over 30minutes and the reaction was left for 6 hours at room temperature. Theflask was maintained at −20° C. for 5 days. More acryoyl chloride (1.5mL) was added. The reaction was left for 24 hours at room temperature.The solvent was removed using a rotary evaporator (the temperature wasset to 30° C.) to produce a white solid. Ethyl acetate (50 mL) was addedand the solid (salt) was collected by filtration. The ethyl acetate wasremoved on the rotary evaporator to leave a yellow oil. Flask columnchromatography using 95% DCM/5% ethyl acetate was used to purify theproduct, increasing to 9/1 ratio then 3/1 ratio. The solvents wereremoved by rotary evaporation before the product was dried at roomtemperature under vacuum.

Yield=0.2176 g (5%)

¹H NMR (CDCl₃) δ ppm: 0.75 (s 2H Me-18) 0.83 (s 2H Me-19) 0.94 (d 3HMe-21) 1-2.43 (steroid structure) 2.67-2.91 (m 2H) 3.60-3.86 (m 1H) 4.63(m 1H CH-3) 5.03 (s 1H CH-7) 5.19 (s 1H CH-12) 5.72-5.97 (m 2H C═CH)5.99-6.52 (m, 3H C═CH)

¹³C NMR (CDCl₃) δ ppm: 174.48 (C═O), 169.70 (C═O), 169.38 (C═O), 169.20(C═O), 165.67 (C═O excess), 165.46 (C═O excess), 165.38 (C═O excess),165.29, 165.23, 136.42 (C═C), 130.55 (C═C), 130.42, 130.26, 130.18,129.12, 129.00, 75.43, 74.62, 74.08, 73.92, 70.94 (C12), 70.85 (C3),51.48, (47.44, 45.23, 45.12, 43.40, 43.32, 43.22, 40.84, 40.74, 39.32,39.20, 39.14, 38.22, 38.00, 37.87, 34.75, 34.62, 34.55, 34.43, 34.35,31.29, 30.91, 30.85, 30.74, 28.82, 28.60, 27.17, 26.74, 26.65 steroidring), 25.46, 25.16, 22.84, 22.5 (C19), 22.34, 17.53 (C21), 17.45, 12.18(C18), 12.08.

MS (+APCI) m/z=Found 602.1935; expected 584.3349 (MI+CH₃+3H)

IR (KBr) ν=2947, 271, 1715, 1633 (C═O), 1612, 1469 cm⁻¹

Synthesis of 3α,12α diacroylate-7αhydroxycholic acid (63)

This product was collected by column chromatography of the previousproduct (3α,7α,12α-triacrylate cholic acid methyl ester).

Yield=0.7744 g (18%)

¹H NMR (CDCl₃) δ ppm: 0.68 (s 2H Me-18) 0.84 (s 2H Me-19) 0.95 (d 3HMe-21) 1-2.43 (Steroid structure) 2.66-2.93 (m 1H) 3.61-3.93 (s 3H CH₃)4.12 (q, J=7.1 Hz, 1H ethyl acetate), 4.64 (tt, J=10.7, 6.5 Hz, 1H CH-3)4.99 (s 1H CH-7) 5.18 (s 1H CH-12) 5.34 (m 1H) 5.70-5.93 (m 1H C═CH)5.98-6.52 (m 2H C═CH)

¹³C NMR (CDCl₃) δ ppm: 174.55 (C═O), (169.68, 169.57, 165.78, 165.71,165.57, 163.18 C═O, including excess acyloyl chloride), (130.66, 130.54,130.30, 130.19, 130.08, 129.12, 129.00, 128.87 C═C, including excessacyloyl chloride), 75.63, 74.40, 74.28, 74.17, 72.57, 71.79, 71.02(C12), 67.93 (C7), 60.36, 51.46, 47.46, 47.11, (46.56, 45.20, 45.06,43.55, 43.40, 42.08, 42.02, 41.18, 41.12, 40.90, 40.84, 39.23, 38.23,38.10, 35.23, 35.16, 34.96, 34.73, 34.61, 34.52, 34.34, 31.31, 30.98,30.81, 28.60, 28.17, 28.10, 27.69, 27.51, 27.24, 26.64 steroid ring),25.51, 25.36, 22.90, 22.53 (C19), 17.41 (C21), 17.32, 12.50, 12.26(C18), 12.19.

MS (+APCI) m/z=Found 548.1638; expected 531.3322 (MI+CH₃+2H)

IR (KBr) ν=35 26 2944, 2871, 1715, 1639 (C═O), 1612, 1469 cm-1

Synthesis of 3α acetate-7α,12α dihydroxy cholic acid (64)

This product was collected by column chromatography of the product3α,7α,12α-triacrylate cholic acid methyl ester.

Yield=0.0232 g

¹H NMR (CDCl₃) δ ppm: 0.70 (s 3H Me-18) 0.82 (s 3H Me-19) 0.94 (d 3HMe-21) 1-2.43 (steroid structure) 3.67 (s 3H CH₃) 3.87 (s 1H CH-7) 4.11(s 1H CH-12) 5.78 (dd J=10.3, 1.7 Hz, 1H C═CH) 6.07 (dd J=17.3, 10.3 Hz1H C═CH) 6.37 (dd J=17.3, 1.7 Hz, 1H)

¹³C NMR (CDCl₃) δ ppm: 217.85, 216.22, 215.83, 215.45, 214.86, 214.47,173.94, 73.08, 71.85 (C12), 68.27 (C7), 56.87, (46.52, 45.30, 41.74,41.49, 40.08, 39.58, 38.83, 35.37, 34.80, 33.50, 33.13, 31.74, 30.41,29.10, 28.16, 27.62, 26.43, 26.10 steroid ring), 25.01, 23.32, 22.69,22.51 (C19), 17.57 (C21), 14.17, 12.50 (C18), 11.48.

MS (+APCI) m/z=Found 494.16; expected 476.3138 (MI+CH₃+3H)

IR (KBr) ν=3375, 2959, 2932, 2865, 1712, 1630 (C═O), 1618, 1533 cm⁻¹

Synthesis of 3α methacrylate 7α,12α dihydroxyl cholic acid methyl ester(65)

Methyl cholate (4.22 g, 10 mmol) was dissolved in dry chloroform (30mL). Triethylamine (2.23 mL, 16 mmol) was added and the flask was put onice. Methacryloyl chloride (1.45 mL, 15 mmol) in chloroform (10 mL) wasdripped in over 30 minutes. The mixture was protected from the light andleft at room temperature overnight. The solvent was removed using arotary evaporator (the temperature was set to 30° C.) to produce a whitesolid. Ethyl acetate (50 mL) was added and the solid (salt) wascollected by filtration. The ethyl acetate was removed on the rotaryevaporator to leave a white solid. Flask column chromatography using 75%DCM/25% ethyl acetate was used to purify the product. The solvents wereremoved by rotary evaporation before the product was dried at roomtemperature under vacuum.

¹H NMR (CDCl₃) δ ppm: 0.70 (s 3H Me-18) 0.83 (s 3H Me-19) 0.98 (d 3HMe-21) 1-2.43 (steroid structure) 3.77 (s 3H CH₃) 3.82 (d, J=26.9 Hz 1HCH-7) 4.10 (s 1H CH-12) 4.63 (tt J=11.2, 4.4 Hz, 1H CH-3) 5.5 (m 1HC═CH) 5.83 (m 1H C═CH) 6.07 (s 1H) 6.25 (s 1H)

¹³C NMR (CDCl₃) δ ppm: 218.61, 174.68 (C═O), 167.10 (C═O), 136.92 (C),124.92 (C═C), 74.54, 72.92 (C12), 68.26 (C7), 51.52, 47.26, (46.57,42.15, 41.23, 39.57, 35.22, 35.13, 34.90, 34.71, 34.42, 31.05, 30.89,28.44, 27.42, 26.83, 26.69 steroid ring), 23.14, 22.57 (C19), 18.35(CH₃), 17.37 (C21), 12.57 (C18).

MS (+APCI) m/z=Found 535.3181; expected 476 (MI+CH₃CN+H₂O)

IR (KBr) ν=3599, 3544, 2971, 2935, 2865, 1733, 1703, 1639 (C═O), 1469cm⁻¹

Synthesis ofN-[2-[1-[(4-vinylphenyl)methyl]pyrrolidin-1-ium-1-yl]ethyl]cholanamidechloride (69)

N-(2-pyrrolidin-1-ylethyl) cholanamide (10) (0.35 g, 0.7 mmol) wasdissolved in chloroform (10 mL). Vinyl benzyl (0.5 mL, 3.5 mmol)chloride was added. The reaction was protected from the light and leftto stir overnight. The reaction was heated under reflux for 24 hours.The solvent was removed under reduced pressure before the product wasdried at room temperature under vacuum.

¹H NMR (CDCl₃) δ ppm: 0.58 (s 3H Me-18) 0.84 (s 3H Me-19) 0.93 (d 3HMe-21) 1-2.43 (steroid ring) 3.11-3.24 (m, 8H 2CH₂), 3.60 (m, 1H CH-3),3.77 (s, 1H CH-7), 3.95 (m, 1H CH-12), 4.35 (d, J=4.0 Hz, 2H CH₂), 4.54(s, 2H CH₂), 5.38 (d, J=10.9 Hz, 1H C═CH), 5.96 (d, J=17.6 Hz, 1H C═CH),6.80 (dd, J=17.7, 11.0 Hz, 1H), 8.23 (d, J=5.2 Hz, 1H NH).

¹³C NMR (DMSO) δ ppm: 173.40 (C═O), (145.12, 138.80, 132.94, 127.83,126.57, 104.54 aromatic ring), 135.74 (C═C), 116.15 (C═C), 79.52, 78.99,78.46, 70.94 (C12), 70.39 (C3), 67.05, 66.21 (C7), 61.14, 60.78, 57.11(CH₂), 48.55, (45.95, 45.72, 41.48, 41.38, 40.50, 40.42, 40.17, 35.28,35.13, 34.86, 34.34, 33.10, 32.23, 31.42, 28.51, 27.30, 26.19 steroidring), 22.58 (C19), 20.86, 17.05 (C21), 14.56, 12.27 (C18).

MS (+APCI) m/z=621.4614

IR (KBr) ν=3350, 3059, 2932, 2862, 1703, 1651 (C═O), 1533 cm⁻¹

Synthesis of 3-acetamidopropyl-dimethyl-[(4-vinylphenyl)methyl]ammoniumchloride (70)

N-[3-dimethylamino) propyl] cholanamide (12) (0.35 g, 0.7 mmol) wasdissolved in chloroform (10 mL). Vinyl benzyl chloride (0.5 mL, 3.5mmol) was added. The reaction was protected from the light and left tostir overnight. The reaction was heated under reflux for 24 hours. Theproduct precipitated out, collected by filtration and washed with petrol40-60 (30 mL). Product not pure.

¹H NMR (CDCl₃) δ ppm: 0.65 (s 3H Me-18) 0.87 (s 3H Me-19) 0.95 (d 3HMe-21) 1-2.43 (steroid structure) 3.49 (s, 1H CH-12), 3.69 (d, J=19.8Hz, 1H CH-7), 3.90 (s, 1H CH-3), 5.38 (dd, J=11.0, 5.9 Hz, 1H CH═CH),5.83 (dd, J=17.7, 4.8 Hz, 1H CH═CH), 6.71 (dd, J=17.6, 10.9 Hz, 1H),7.30-7.63 (m, 4H aromatic ring), 8.49 (s, 1H NH)

¹³C NMR (DMSO) δ ppm: 172.97 (C═O), (138.80, 135.76, 133.21, 127.38,126.40 aromatic ring), 116.09 (C═C), 79.52, 78.99, 78.46, 70.97 (C12),70.39 (C3), 66.20 (C7), 65.85, 61.35, 59.67 (CH₃), 49.22, 48.55, (45.92,41.48, 41.36, 40.51, 40.18, 35.51, 35.27, 35.18, 34.87, 34.35, 32.31,28.51, 27.28, 26.20 steroid ring), 45.70 (CH₃), 31.50 (CH₂), 22.79,22.68, 22.58 (C19), 17.11 (C21), 14.61, 12.29 (C18).

MS (+APCI) m/z=609.4619

IR (KBr) ν=3347, 2929, 2862, 1775, 1694, 1627 (C═O), 1551 cm⁻¹

Synthesis ofN-[1-phenyl-1-[(4-vinylphenyl)methyl]piperidin-1-ium-4-yl]cholanamidechloride (71)

N-(1-phenyl-4-piperidyl) cholanamide (13) (1.5 g, 3.5 mmol) wasdissolved in DCM (20 mL). Vinyl benzyl chloride (1.5 mL, 10.5 mmol) wasadded. The reaction was protected from the light and left to stirovernight. The reaction was heated under reflux for 24 hours. Theproduct precipitated out, which was then collected by filtration andwashed with petrol 40-60 (30 mL). Further purification by solventextraction (×3) between petrol 40-60 (10 mL) and methanol (10 mL) left awhite solid.

¹H NMR (CDCl₃) δ ppm: 0.57 (s32H Me-18) 0.88 (s 3H Me-19) 0.98 (d 3HMe-21) 1-2.43 (steroid structure) 3.18 (tt, J=14.7, 7.3 Hz, 7H CH₂ring), 3.61 (d, J=3.7 Hz, 1H CH-3), 3.77 (d, J=3.6 Hz, 1H CH-7), 4.04(d, J=3.2 Hz, 1H CH-12), 4.14 (d, J=3.3 Hz, 1H C3-OH), 4.36 (d, J=4.0Hz, 1H C7-OH), 4.53 (s, 1H C12-OH), 5.38 (d, J=10.9 Hz, 1H CH═CH), 5.95(d, J=17.7 Hz, 1H CH═CH), 6.79 (dd, J=17.6, 11.0 Hz, 1H), 7.73-7.43 (m,4H aromatic ring), 8.07 (t, J=5.7 Hz, 1H NH).

¹³C NMR (DMSO) δ ppm: 172.94 (C═O), (138.80, 135.78, 133.22, 127.43,126.42, aromatic ring), 116.15 (C═C), 70.95 (C12), 70.37 (C3), 66.19(C7), 65.83, 61.33, 49.24, (45.94, 45.70, 41.48, 41.36, 40.51, 40.17,35.50, 35.28, 35.19, 34.87, 34.35, 32.32, 31.51, 30.35, 28.54, 27.29,26.20 steroid ring), 22.79, 22.67, 22.60 (C19), 17.12 (C21), 12.31(C18).

MS (+APCI) m/z=Found 609.4612; expected 683.4782 (MI-73)MI−(vbc+propane)

IR (KBr) ν=3356, 2925, 2862, 2164, 1648 (C═O), 1548 cm⁻¹

Synthesis of N-Boc-1,4-butanediamine (72)

1,4-diaminobutane (1 mL, 12.94 mmol) was dissolved in DCM (20 mL) andput on ice. Di-tert-butyl dicarbonate (0.3 g, 1.29 mmol) in DCM (5 mL)was dripped in over 50 minutes. The reaction was left at ice temperaturefor 12 hours. The reaction was washed with water (100 mL) followed bybrine (100 mL), before being dried with magnesium sulphate. The solventwas removed under vacuum to leave a yellow solid.

Yield=0.087 g (8.7%)

¹H NMR (CDCl₃) δ ppm: 1.40-1.61 (m 12H) 3.13 (q J=6.1 Hz 2H) 4.55 (s 1H)

¹³C NMR (DMSO) δ ppm: 155.57 (C═O), 77.26 (C), 28.36 (CH₃), 28.21,27.29, 26.93, 26.86.

MS (+APCI) m/z=Found 289.2126; expected 188. Diamer produced.

IR (KBr) ν=3372 2983 2941 2847 1681 (C═O) 1521 cm⁻¹

Polymerisation of N-(4-cholanamidobutyl)-2-methyl-prop-2-enamide

N-(4-cholanamidobutyl)-2-methyl-prop-2-enamide (75) (0.6 g, 0.96 mmol)was dissolved in methanol (7 mL). Styrene (2.4 mL, 23.04 mmol) was addedalong with a small amount of AIBN. The boiling tube was purged withargon 3 times and put into an oil bath at 60° C. for 48 hours. The whitesolid precipitate was collected by filtration, dissolved into chloroformand dripped onto stirring methanol (50 mL) for purification. The polymerwas collected by filtration, washed with methanol (30 mL) and dried atroom temperature under vacuum.

Polymerisation of 3α,12α diacroylate-7αhydroxycholic acid

3α,12α diacroylate-7αhydroxycholic acid (63) (0.175 g) was dissolved intoluene (2.5 mL). AIBN (0.1 g) was added, along with EGDMA (0.82 mL).1000 of the solution was pipetted out into a 96 well plate and aPTFE-lined cover was clamped across the top of the plate to seal thewells. It was place in at oven set at 60° C. for 12 hours. The productswere white discs.

Polymerisation of methacrylate 7α,12α dihydroxyl cholic acid

Methacrylate 7α,12α dihydroxyl cholic acid (65) (0.2 g) was dissolved intoluene (3.5 mL). AIBN (0.1 g) was added, along with EGDMA (0.8 mL).1000 of the solution was pipetted out into a 96 well plate and clamped.It was place in at oven set at 60° C. for 12 hours. The products werewhite discs.

Polymerisation of 3α hydroxyl 7α,12α diacetate methyl cholate 3αhydroxyl 7α,12α diacetate methyl cholate (77) (0.5 g) was added to around bottomed flask along with bis[acetylacetonato]copper (0.02 g) andvinyl benzyl chloride (2 mL). The reaction was heated to 120° C. for 4.5hours. The crude product was purified using column chromatography withpetrol 60-80, slowly increasing the amount of ethyl acetate (0-100%).The solvents were removed under reduced pressure and the polymer wasdried at room temperature under vacuum.

Polymerisation of3-acetamidopropyl-dimethyl-[(4-vinylphenyl)methyl]ammonium chloride (70)

3-acetamidopropyl-dimethyl-[(4-vinylphenyl)methyl]ammonium chloride (70)(0.5 g) was dissolved in ethanol (4 mL). Styrene (3.9 mL) was addedalong with AIBN (100 mg). The reaction was degassed and heated to 65° C.for 12 hours where a white solid precipitated out. It was purified bydissolving in chloroform (20 mL) and dripping into stirring methanol(100 mL). The product was collected by filtration and dried at roomtemperature under vacuum to give a white solid.

Proton NMR analysis shows very little incorporation ofN-[3-(dimethylamino)propyl]cholanamide (12) into the polymer.

3-acetamidopropyl-dimethyl-[(4-vinylphenyl)methyl]ammonium chloride(70)(0.25 g) was dissolved in ethanol (4 mL) along with AIBN (100 mg).Tert-butyl methacrylate (0.23 mL) was added. The mixture was degassedwhich argon and heated to 60° C. for 4 days. The solution was purifiedby dripping onto ethyl acetate (75 mL) to give a white powdery solidwhich was dried at room temperature under vacuum.

Proton NMR analysis showed that no polymerisation had taken place.

3-acetamidopropyl-dimethyl-[(4-vinylphenyl)methyl]ammonium chloride (70)(0.25 g) was suspended in propan-2-ol (6 mL) along with AIBN (100 mg).The mixture was degassed and heated to 70° C. for 5 days. The white,cloudy solution was collected by filtration to give a off white solidwhich was dried at room temperature under vacuum.

No NMR analysis could be undertaken of this polymer due to itsinsolubility.

Polymerisation of Synthesis ofN-[1-phenyl-1-[(4-vinylphenyl)methyl]piperidin-1-ium-4-yl]cholanamidechloride (71)

Synthesis ofN-[1-phenyl-1-[(4-vinylphenyl)methyl]piperidin-1-ium-4-yl]cholanamidechloride (70) (0.3 g) was dissolved in proan-2-ol (8 mL) along with AIBN(100 mg). The mixture was degassed with argon and heated to 70° C. for 6days. An white solid precipitated out, which was collected by filtrationand dried at room temperature under vacuum.

Proton NMR analysis shows a very short chained polymer was synthesised.

Synthesis of 4-benzylbenzoyl chloride (96)

4-benzyl benzoic acid (1 g, 4.42 mmol) was added to a dry flask alongwith thionyl chloride (3 mL), DMF (0.5 mL) and toluene (13 mL). Theflask was heated under reflux for 5 days. The solvent was removed underreduced pressure and the product was re-dissolved in toluene (5 mL)twice and the solvent removed. The product was dried under vacuum atroom temperature under vacuum to give a white solid.

Yield=1.03 g

¹H NMR (CDCl₃) δ ppm: 8.56, 8.54, 8.29, 8.24, 8.24, 8.23, 8.21, 8.20,8.20, 8.19, 8.18, 7.93, 7.90, 7.89, 7.88, 7.87, 7.86, 7.86, 7.85, 7.84,7.82, 7.81, 7.80, 7.80, 7.78, 7.78, 7.77, 7.67, 7.66, 7.65, 7.64, 7.63,7.62, 7.62, 7.61, 7.60, 7.60, 7.59, 7.58, 7.54, 7.53, 7.52, 7.50, 7.50,7.49, 7.48, 7.47, 7.47, 7.24, 6.82, 3.95, 3.47, 3.33, 2.15, 1.79,0.217.83-7.73 (m, 2H), 7.96-7.80 (m, 2H), 8.30-8.13 (m, 2H),

¹H NMR (250 MHz, Chloroform-d) δ 7.69-7.56 (m, 1H), 7.50 (tt, J=6.6, 1.5Hz, 2H).

MS (+APCI) m/z=Found 245.0366; calculated for C₁₄H₁₀Cl₁O₂ 245.0364; 0.9ppm

Synthesis of N-(4-benzoylphenyl)formamide cholate (98)

Cholic acid (0.5 g, 1.2 mmol) was dissolved in THF (30 mL) along withtriethylamine (2.9 mL, 0.3 mmol). The solution was put on ice for 10minutes before ethylchloroformate (0.13 mL, 0.013 mmol) was dripped inover 10 minutes. The solution was allowed to react for two hours at roomtemperature. 4-aminobenzophenone (0.23 g, 1.2 mmol) was added and leftto react for 3 hours. The reaction was quenched with water (30 mL). Themixture was washed with water (3×30 mL). The organic layer was driedover magnesium sulphate and the solvent was evaporated under reducedpressure. Solvent extraction between water and ethyl acetate waspreformed 3 times before the organic layer was removed under reducedpressure. The product was dried at room temperature under vacuum.

MS (+APCI) m/z=Found 588.3680; calculated for C₃₇H₅₀N₁O₅ 588.3684; −0.6ppm

Synthesis of methyl lithocholate (99)

Lithocholic acid (5.0 g, 0.01329 mol) was added to methanol (90 mL) toproduce a suspension. Acetyl chloride (0.5 mL, 0.006 mol) was thenadded. The solution was heated and stirred at 80° C. for 40 minutes as ahomogeneous solution, then allowed to cool overnight in an ice bath.This was added to water (150 mL) and the resulting precipitate wascollected by filtration, washed with water (3×20 mL) and dried undervacuum.

Yield; 5 g, 0.01328 mol, 96.5%.

Melting point: 75-76° C.

¹H NMR (250 MHz) δ=0.66 (s, 3H, 18-CH₃), 0.94 (s, 3H, 19-CH₃), 3.65 (m,1H, 3-CH), 3.69 (s, 3H, O—CH₃) ppm.

¹³C NMR (62.9 MHz) δ=12.0 (CH₃, C18), 18.6 (CH₃, C21), 20.8 (CH₂, C11),23.7 (CH₃, C19), 24.2 (CH₂, C15), 26.4 (CH₂, C7), 27.2 (CH₂, C6), 28.2(CH₂, C16), 30.5 (CH₂, C2), 30.9 (CH₂, C22), 31.0 (CH₂, C23), 34.5 (C,C10), 35.3 (CH, C20), 35.4 (CH₂, C1), 35.8 (CH, C8), 36.4 (CH₂, C4),40.1 (CH₂, C12), 40.4 (CH, C9), 42.0 (CH, C5), 42.7 (C, C13), 51.5(O—CH₃) 55.9 (CH, C17), 56.5 (CH, C14), 71.9 (CH, C3), 174.8 (CO, C24)ppm.

IR; 3347 (OH stretch), 2937 (C—H), 2859 (O—CH₃), 1733 (C═O), 1640, 1436,1206, 1043 (R₂CH—OH) cm⁻¹.

Synthesis of methyl deoxycholate (100)

Deoxycholic acid (5.0 g 0.01 mol) was dissolved in methanol (30 mL) andtreated with acetyl chloride (0.5 mL, 0.006 mol). The solution washeated and stirred at 80° C. for 40 minutes then allowed to coolovernight in an ice bath. The resultant crystals were collected byvacuum filtration to produce a white crystalline powder. This was washedwith water (2×20 mL) and dried under vacuum.

Yield; 1.81 g, 0.004 mol, 35%.

Melting point: 70-72° C.

¹H NMR (250 MHz) δ=0.68 (s, 3H, 18-CH₃), 0.91 (s, 3H, 19-CH₃), 3.60 (m,1H, 3-CH), 3.67 (s, 3H, O—CH₃), 3.99 (s, 1H, 12-CH) ppm.

¹³C NMR (62.9 MHz) δ=12.8 (CH₃, C18), 17.3 (CH₃, C21), 23.2 (CH₃, C19),26.6 (CH₂, C15), 26.1 (CH₂, C7), 27.1 (CH₂, C6), 27.4 (CH₂, C16), 28.7(CH₂, C11), 30.5 (CH₂, C2), 30.9 (CH₂, C23), 31.1 (CH₂, C22), 33.7 (CH,C9), 34.1 (C, C10), 35.1 (CH₂, C1), 35.2 (CH, C20), 36.0 (CH₂, C4), 36.4(CH, C8), 42.0 (CH, C5), 46.5 (C, C13), 47.3 (CH, C17), 48.3 (CH, C14),51.5 (O—CH₃) 71.8 (CH, C3), 73.1 (CH₂, C12), 174.7 (CO, C24) ppm.

IR; 3474 (OH stretch) 2985 (C—H), 2852 (O—CH₃), 1743 (C═O), 1450, 1380,1040 (R₂CH—OH) cm⁻¹.

¹³C NMR (62.9 MHz) δ=12.0 (CH₃, C18), 18.2 (CH₃, C21), 20.8 (CH₂) 23.3(CH₃, C19), 24.2 (CH₂, C15), 26.4 (CH₂, C7), 27.1 (CH₂, C6), 28.2 (CH₂,C16), 30.5 (CH₂, C11), 30.9 (CH₂, C2), 31.1 (CH₂, C23), 34.5 (CH, C9),35.3 (C, C10), 35.8 (CH₂, C1), 36.4 (CH, C20), 40.1 (CH, C5), 40.4 (C,C13), 55.9 (CH₂), 56.5 (CH₂), 61.3 (CH₂) 65.9 (CH, C3), 71.8 (CH₂, C12),174.7 (CO, C24) ppm.

IR=3509 (OH), 3309 (OH), 2925 (alkyl), 2857 (alkyl), 1718 (C═O), 1449,1356, 1292, 1189.78 (C═O ester stretch), 1027.73 (R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 454.3527 (M+H)⁺; calculated for C₂₆H₄₈NO₅ 454.3527;0.0 ppm.

Synthesis of(4R)—N-(2-dimethylaminoethyl)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanamide(101)

A mixture of lithocholic acid (1.0 g 0.005 mol) and N,N′-dimethylethylenediamine (0.16 mL 0.001 mol) was dissolved in toluene(20 mL). The solution was heated at reflux for 24 hours. More N,N′-dimethylethylenediamine (0.32 mL 0.003 mol) of was added and themixture was heated at reflux for 24 h. Water (100 mL) was added to thesolution. The resulting precipitate was collected by vacuum filtrationand the crude product was recrystallized from ethyl acetate to produce awhite powder.

Yield 0.85 g, 0.001 mol, 69%.

¹H NMR (250 MHz) CDCl₃ δ=0.64 (s, 3H, 18-CH₃), 0.92 (s, 3H, 19-CH₃),2.21 (s, 6H, 2×CH₃), 2.42 (t, 2H CH₂ J=5.0) 3.34 (q, 2H, CH₂, J=7.5),3.63 (m, 1H, 3-CH), 6.20 (broad s, 1H, NH) ppm.

¹³C NMR (62.9 MHz) δ=12.0 (CH₃, C18), 18.3 (CH₃, C21), 20.8 (CH₂, C11),23.3 (CH₃, C19), 24.2 (CH₂, C15), 26.4 (CH₂, C7), 27.2 (CH₂, C6), 28.2(CH₂, C16), 30.5 (CH₂, C2), 31.7 (CH₂, C22), 33.5 (CH₂, C23), 34.5 (C,C10), 35.3 (CH, C20), 35.5 (CH₂, C1), 35.8 (CH, C8), 36.4 (CH₂, C4),36.6 (CH₂) 40.2 (CH₂, C12), 40.4 (CH, C9), 42.1 (CH, C5), 42.7 (C, C13),45.1 (CH₃) 56.0 (CH, C17), 56.5 (CH, C14), 57.9 (CH₂) 71.8 (CH, C3),173.7 (CO, C24) ppm.

IR; 3377 (OH), 3293 (NH) 2929 (C—H), 2870 (C—H), 1646 (C═O), 1543, 1445cm⁻¹.

MS (+ESI) m/z=Found 447.3943 (M+H)⁺; calculated for C₂₈H₅₁N₂O₂ 447.3945;0.5 ppm.

Synthesis of(4R)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-N-(2-dimethylaminoethyl)pentanamide(102)

A mixture of deoxycholic acid (1.0 g 0.002 mol) and N,N′-dimethylethylenediamine (0.16 mL 0.001 mol) was dissolved in toluene(20 mL). The solution was heated at reflux for 24 hours. More N,N′-dimethylethylenediamine (0.16 mL 0.001 mol) was added and thereaction mixture was heated at reflux for a further 24 hours. Thesolvent was then evaporated under reduced pressure. The residue wastriturated with hot water (˜10 mL) and washed with water (3×20 mL), thendried under vacuum.

Yield 0.310 g, 0.0006 mol, 24%.

¹H NMR (CDCl₃) (250 MHz) δ=0.68 (s, 3H, 18-CH₃), 0.91 (s, 3H, 19-CH₃),1.01 (d, 3H, 21-CH₃, J=6.5), 2.26 (s, 6H, 2×CH₃), 2.46 (t, 2H, CH₂,J=5.0), 3.34 (t, 2H, CH₂, J=5.0), 3.61 (m, 1H, 3-CH), 3.97 (broadsinglet, 1H, 12-CH), 6.44 (broad singlet, 1H, NH) ppm.

¹³C NMR (62.9 MHz) δ=12.7 (CH₃, C18), 17.5 (CH₃, C21), 23.1 (CH₃, C19),23.6 (CH₂, C15), 26.1 (CH₂, C7), 27.1 (CH₂, C6), 27.8 (CH₂, C16), 28.5(CH₂, C11), 30.5 (CH₂, C2), 31.6 (CH₂, C23), 33.3 (CH₂, C22), 33.6 (CH,C9), 34.1 (C, C10), 35.2 (CH₂, C1), 36.0 (CH, C20), 36.4 (CH₂, C4), 42.0(CH, C5), 44.9 (C, C13), 46.5 (CH₃) 47.3 (CH, C17), 48.2 (CH, C14), 57.9(CH₃) 71.7 (CH, C3), 73.0 (CH₂, C12), 172.7 (CO, C24) ppm.

IR=3305, (OH), 2929 (alkyl), 2861 (alkyl), 1720 (C═O), 1044 (R₂CH—OH)cm⁻¹

MS (+ESI) m/z=Found 463.3888 (M+H)⁺; calculated for C₂₈H₅₁N₂O₃ 463.3894;1.3 ppm.

Synthesis of(4R)—N-[3-(dimethylamino)propyl]-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanamide(103)

A mixture of methyl lithocholate (0.5 g 0.001 mol) and3-dimethyl-propylamine (3 mL, 0.02 mol) was heated and stirred at 140°C. for 24 hours in an argon environment. Ice water (3 mL) was added tothe material and left to stir for two hours at room temperature. Theresulting solid was then collected by filtration washed with water (3×20mL) and left to dry overnight under vacuum to produce off browncrystals. Yield, 0.41 g, 0.0008 mol, 69%.

Melting point: 177.0-178.3° C.

¹H NMR CDCl₃ (250 MHz) δ=0.61 (s, 3H, 18-CH₃) 0.89 (d, 3H, 19-CH₃) 1.57(s, 3H, 21-CH₃) 2.22 (s, 6H, 2×CH₃) 2.36 (t, 2H, CH₂, J=6.3) 3.30 (t,2H, CH₂, J=5.6) 3.59 (m, 1H, 3-CH) 6.95 (s, 1H, NH) ppm.

¹³C NMR (62.9 MHz) δ=12.0 (CH₃, C18), 18.3 (CH₃, C21), 20.8 (CH₂, C11),23.3 (CH₃, C19), 24.2 (CH₂, C15), 24.3 (CH₂, C7), 26.4 (CH₂), 27.1 (CH₂,C6), 28.2 (CH₂, C16), 30.5 (CH₂, C2), 31.7 (CH₂, C22) 34.5 (C, C10),35.3 (CH, C20), 35.5 (CH₂, C1), 35.8 (CH, C8), 36.4 (CH₂, C4), 40.1(CH₂, C12), 40.3 (CH, C9), 42.0 (CH, C5), 42.7 (C, C13), 43.0 (CH₃) 55.3(CH, C17), 55.9 (CH, C14), 56.4 (CH₂) 71.9 (CH, C3), 174.9 (CO, C24)ppm.

IR=3310-3318 (OH—NH), 2730 (alkyl), 2859 (alkyl), 2946 (alkyl), 1648(C═O), 1047 (CH—OH) cm⁻¹.

MS (ES+APCI) m/z=Found 461.4105 (M+H)⁺; calculated for C₂₉H₅₃N₂O₂461.4107; 0.4 ppm.

Synthesis of(4R)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-N-[3-(dimethylamino)propyl]pentanamide(104)

A mixture of methyl deoxycholate (0.5 g 0.002 mol) and3-dimethyl-1-propylamine (3.63 mL 0.03 mol) were added together andheated to 100° C. for 5 days then allowed to cool to ambient roomtemperature. Ice water (40 mL) was then added and left to stir for 2hours and the precipitate collected by vacuum filtration, washed withwater (3×20 mL) and dried under vacuum.

Yield, 0.41 g, 0.0008 mol, 73%.

Melting point: 123-127° C.

¹H NMR (CDCl₃) (250 MHz) δ=0.64 (s, 3H, 18-CH₃), 0.92 (s, 3H, 19-CH₃),2.23 (s, 6H, 2×CH₃), 2.42 (t, 2H, CH₂, J=5.0), 3.325 (q, 2H, CH₂,J=7.5), 3.63 (m, 1H, 3-CH), 6.21 (broad singlet, 1H, NH) ppm.

¹³C NMR (62.9 MHz) δ=12.0 (CH₃, C18), 18.3 (CH₃, C21), 20.8 (CH₂, C11),23.3 (CH₃, C19), 24.2 (CH₂, C15), 26.4 (CH₂, C7), 27.2 (CH₂, C6), 28.2(CH₂, C16), 30.5 (CH₂, C2), 31.7 (CH₂, C22), 33.5 (CH₂, C23), 34.5 (C,C10), 35.3 (CH, C20), 35.5 (CH₂, C1), 35.8 (CH, C8), 36.4 (CH₂, C4),36.6 (CH₂), 40.2 (CH₂, C12), 40.4 (CH, C9), 42.1 (CH, C5), 42.7 (C,C13), 45.1 (CH₂), 56.0 (CH, C17), 56.5 (CH₂), 57.9 (CH, C14), 71.8 (CH,C3), 173.7 (CO, C24) ppm.

IR=3376 (NH and OH), 2938 (alkyl), 2861 (alkyl), 1643 (C═O), 1544, 1444,1378, 1045 (R₂CH—OH) cm⁻¹.

MS (+ES APCI) m/z=Found 447.3943 (M+H)⁺; calculated for C₂₈H₅₁N₂O₂447.3943; 0.5 ppm.

Synthesis of1-(4-butylpiperazin-1-yl)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentan-1-one(105)

Following a modified method (Fini et al., 1992), lithocholic acid (0.5 g0.001 mol) was dissolved in tetrahydrofuran (20 mL) with triethylamine(0.4 mL 0.002 mol). The solution was cooled for 10 minutes in coldwater. Ethyl chloroformate (0.2 mL 0.001 mol) was then added dropwiseover a ten minute period. Once added, the cold water was removed and thesolution was stirred for 2 hours. After 2 hours, solvent removed underreduced pressure then re-dissolved in dichloromethane and washed withwater then 1-butylpiperazine (0.25 mL 0.001 mol) was added and thesolution was stirred for 24 hours. Water (50 mL) was then added and thesolution extracted with ethyl acetate (3×50 mL). The organic layers werecombined and washed with saturated sodium hydrogen carbonate solution(3×50 mL). The organic layer was dried over magnesium sulphate. Solventwas evaporated under reduced pressure.

Yield; 0.16 g, 0.0003 mol, 23.5%.

Melting point: 172.7-173.1° C.

¹H NMR (CDCl₃) (250 MHz) δ=0.61 (s, 3H, 18-CH₃), 0.88 (s, 3H, 19-CH₃),1.34 (m, 4H, CH₂), 2.35 (m, 6H, CH₂), 3.45 (broad singlet, 2H, CH₂),3.60 (broad singlet, 2H, CH₂) ppm.

¹³C NMR (62.9 MHz) δ=12.0 (CH₃, C18), 14.0 (CH₃), 18.5 (CH₃, C21), 20.6(CH₂, C11), 20.8 (CH₂) 23.3 (CH₃, C19), 24.2 (CH₂, C15), 26.4 (CH₂, C7),27.2 (CH₂, C6), 28.2 (CH₂, C16), 28.8 (CH₂), 30.2 (CH₂, C2), 30.5 (CH₂,C22), 31.4 (CH₂, C23), 34.5 (C, C10), 35.3 (CH, C20), 35.6 (CH₂, C1),35.8 (CH, C8), 36.4 (CH₂, C4), 40.1 (CH₂, C12), 40.4 (CH, C9), 41.4 (CH,C5), 42.1 (C, C13), 42.8 (CH₂), 45.6 (CH₂), 52.9 (CH₂), 53.4 (CH₂), 53.4(CH₂), 56.0 (CH, C17), 56.5 (CH, C14), 58.3 (CH₂), 71.7 (CH, C3), 172.1(CO, C24) ppm.

IR=3381, (OH), 2916 (alkyl), 2852 (alkyl), 1616 (C═O), 1437, 1253, 1036(R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 501.4409 (M+H)⁺; calculated for C₃₂H₅₇N₂O₂ 501.4409;1.1 ppm.

Synthesis of(4R)-1-(4-butylpiperazin-1-yl)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentan-1-one(106)

Deoxycholic acid (0.5 g 0.001 mol) was dissolved in tetrahydrofuran (20mL) with triethylamine (0.4 mL 0.001 mol). The solution was cooled for10 minutes in cold water. Ethyl chloroformate (0.2 mL 0.001 mol) wasthen added dropwise over a ten minute period. Once added, the cold waterwas removed and the solution was stirred for 2 hours. After 2 hours,solvent removed under reduced pressure then re-dissolved indichloromethane and washed with water then 1-butylpiperazine (0.25 mL0.001 mol) was added and the solution was stirred for 24 hours. Water(50 mL) was then added and the solution extracted with ethyl acetate(3×50 mL). The organic layers were combined and washed with saturatedsodium hydrogen carbonate solution (3×50 mL). The organic layer wasdried over magnesium sulphate. Solvent was evaporated under reducedpressure.

Yield; 0.04 g, 0.0004 mol, 6%.

Melting point: 92.3-93.5° C.

¹H NMR (CDCl₃) (250 MHz) δ=0.68 (s, 3H, 18-CH₃), 0.89 (s, 3H, 19-CH₃),0.98 (d, 3H, 21-CH₃ J=7.5), 2.38 (m, 6H, CH₂), 3.48 (broad singlet, 2H,CH₂), 3.62 (broad singlet, 2H, CH₂), 3.98 (broad singlet, 1H, 12-CH)ppm.

¹³C NMR (62.9 MHz) δ=12.7 (CH₃, C18), 14.0 (CH₂), 17.5 (CH₃, C21), 20.6(CH₂), 23.1 (CH₃, C19), 23.6 (CH₂, C15), 27.5 (CH₂, C7), 28.6 (CH₂, C6),28.8 (CH₂, C16), 30.1 (CH₂, C11), 30.5 (CH₂, C2), 31.3 (CH₂, C23), 33.6(CH₂, C22), 34.1 (CH, C9), 35.2 (C, C10), 35.2 (CH₂, C1), 36.0 (CH,C20), 36.4 (CH₂, C4), 41.4 (CH₂), 42.0 (CH, C5), 45.6 (CH₂) 46.5 (CH₂)47.2 (CH, C17), 48.3 (CH, C14), 52.8 (CH₂) 53.4 (CH₂) 71.8 (CH, C3),73.1 (CH₂, C12), 171.9 (CO, C24) ppm.

IR=3300 (OH), 2930 (alkyl), 2861 (alkyl), 1594 (C═O), 1443, 1282, 1162(C═O ester stretch), 1011 (R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 517.4359 (M+H)+; calculated for C₃₂H₅₇N₂O₃ 517.4364;0.9 ppm.

Synthesis of4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-N-(2-pyrrolidin-1-ylethyl)pentanamide(107)

Lithocholic acid (0.5 g 0.001 mol) was dissolved in tetrahydrofuran (20mL) with triethylamine (0.20 mL, 0.001 mol). The solution was cooled for10 minutes in cold water. Ethyl chloroformate (0.20 mL, 0.001 mol) wasthen added dropwise over a ten minute period. Once added, the cold waterwas removed and the solution was stirred for 2 hours at roomtemperature. After 2 hours 1-(2-aminoethyl)pyrollidine (0.25 mL, 0.002mol) was added and the solution was stirred for 3 hours. Water (50 mL)was then added and the solution was extracted with ethyl acetate (3×50mL). The organic layers were combined and dried over magnesium sulphate.The solvent was evaporated under reduced pressure to produce a whitepowder.

Yield; 0.56 g, 0.001 mol, 91.8%.

Melting point: 144.1-145.1° C.

¹H NMR (CDCl₃) (250 MHz) δ=0.64 (s, 3H, 18-CH₃), 0.92 (s, 3H, 19-CH₃)0.94 (d, 3H, 21-CH₃, J=7.5), 1.80 (m, 4H, CH₂), 2.54 (broad singlet, 4H,CH₂), 2.60 (t, 2H, CH₂, J=5.0) 3.63 (q, 2H, CH₂, J=5.0) 3.63 (m, 1H,3-CH), 6.13 (broad s, 1H, NH) ppm.

¹³C NMR (62.9 MHz) δ=12.0 (CH₃, C18), 18.4 (CH₃, C21), 20.8 (CH₂, C11),23.4 (CH₃, C19), 24.2 (CH₂, C15), 26.4 (CH₂, C7), 27.2 (C H₂, C6), 28.2(CH₂, C16), 30.5 (CH₂, C2), 31.7 (CH₂, C22), 33.5 (CH₂, C23), 34.5 (C,C10), 35.3 (CH, C20), 35.5 (CH₂, C1), 35.8 (CH, C8), 36.4 (CH₂, C4),37.7 (CH₂) 40.2 (CH₂, C12), 40.4 (CH, C9), 42.1 (CH, C5), 42.7 (C, C13),53.8 (CH₂), 54.9 (CH₂) 56.0 (CH, C17), 56.5 (CH, C14), 71.8 (CH, C3),173.9 (CO, C24) ppm.

IR=3415 (NH), 3310 (OH), 2933 (alkyl), 2865 (alkyl), 2872 (alkyl), 1648(C═O), 1548, 1444, 1378, 1265 (C═O ester stretch), 1064 (R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 473.4096 (M+H)⁺; calculated for C₃₀H₅₃N₂O₂ 473.4102;1.2 ppm.

Synthesis of4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-N-(2-pyrrolidin-1-ylethyl)pentanamide(108)

Deoxycholic acid (2.0 g 0.005 mol) was dissolved in dioxane (60 mL) withtriethylamine (1.28 mL, 0.01 mol). The solution was cooled for 10minutes in cold water. Ethyl chloroformate (0.37 mL, 0.003 mol) was thenadded dropwise over a ten minute period. Once added, the cold water wasremoved and the solution was stirred for 2 hours. After 2 hours1-(2-aminoethyl)pyrollidine (0.77 mL, 0.006 mol) was added and thesolution was stirred for 3 hours at room temperature. Water (50 mL) wasthen added and the solution was extracted with ethyl acetate (3×50 mL).The organic layers were combined and washed with saturated sodiumhydrogen carbonate solution (3×50 mL). The organic layer was dried overmagnesium sulphate. The solvent was evaporated under reduced pressure toproduce a white powder.

Yield; 1.2 g, 0.0025 mol, 49%.

Melting point: 155.3-158.7° C.

¹H NMR (CDCl₃) (250 MHz) δ=0.66 (s, 3H, 18-CH₃), 0.89 (s, 3H, 19-CH₃),0.975 (d, 3H, 21-CH₃, J=7.5), 1.78 (m, 4H, CH₂), 2.42-2.58 (m, 6H, CH₂),3.35 (m, 2H, CH₂), 3.58 (m, 1H, 3-CH), 3.95 (broad s, 1H, 12-CH), 6.49(broad s, 1H, NH) ppm.

¹³C NMR (62.9 MHz) δ=12.7 (CH₃, C18), 17.5 (CH₃, C21), 23.3 (CH₃, C19),23.7 (CH₂, C15), 26.1 (CH₂, C7), 27.1 (CH₂, C6), 28.6 (CH₂, C16), 30.4(CH₂, C11), 30.5 (CH₂, C2), 33.3 (CH₂, C23), 33.6 (CH₂, C22), 34.1 (CH,C9), 35.2 (C, C10), 35.3 (CH₂, C1), 36.0 (CH, C20), 36.5 (CH₂, C4), 37.9(CH, C8), 42.1 (CH₂), 46.5 (C, C13), 46.8 (CH, C17), 48.2 (CH, C14),53.9 (CH), 55.0 (CH₂) 71.5 (CH, C3), 73.0 (CH₂, C12), 173.9 (CO, C24)ppm.

IR=3287 (OH), 2916 (alkyl), 2865 (alkyl), 1641 (C═O), 1539, 1441, 1040(R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 489.4046 (M+H)⁺; calculated for C₃₀H₅₃N₂O₃ 489.4051;1 ppm.

Synthesis of4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-N-[2-(1-piperidyl)ethyl]pentanamide(109)

Methyl lithocholate 0.5 g (0.001 mol) 1,2 amino-ethyl piperidine 0.36 mL(0.002 mol) were heated at and stirred at 150° C. for 48 hours in aargon environment. More 1,2 amino-ethyl piperidine (1 mL, 0.007 mol) ofwas added and the reaction was continued under the same conditionsovernight. The crude material was purified by flash columnchromatography (1:1 EtOAc/MeOH) to give a solid.

Yield; 0.01 g, 0.00001 mol, 1.6%.

Melting point: 85.0-89.9° C.

¹H NMR CDCl₃ (250 MHz) δ=0.59 (s, 3H, 18-CH₃) 0.87-0.89 (s, 3H, 19-CH₃J=5.5) 1.72 (m, 4H, CH₂) 2.68 (t, 2H, CH₂, J=5.6) 3.42-3.44 (q, 2H, CH₂,J=5.6) 3.58 (m, 1H, 3-CH) 7.18 (s/t, 1H, NH) ppm.

¹³C NMR (62.9 MHz) δ=18.3 (CH) 23.4 (CH₃) 24.2 (CH₃CH) 26.4 (CH₂) 42.2(CH₃) 56.5 (CH₂) 71.8 (COH) 173.9 (CO) ppm.

IR=3418 (NH), 3465 (OH) 2935 (alkyl), 2865 (alkyl), 1641 (C═O) cm⁻¹.

MS (ES) m/z=Found 487.4347 (M+H)⁺; calculated for C₃₁H₅₄N₂O₃ 487.4264;0.8 ppm.

Synthesis of(4R)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-N-[2-(1-piperidyl)ethyl]pentanamide(110)

Methyl deoxycholate (1.0 g 0.002 mol) was dissolved in methanol (10 mL)with 1-(2-aminoethyl)piperidine (3.15 g, 0.024 mol) was added and thesolution was stirred for 5 days. The solvent was evaporated underreduced pressure and the product was recrystallized from ethyl acetateto produce a white powder.

Yield; 0.56 g, 0.0011 mol, 45%.

Melting point: 158.5-161.1° C.

¹H NMR (CDCl₃) (250 MHz) δ=0.66 (s, 3H, 18-CH₃), 0.90 (s, 3H, 19-CH₃),0.99 (d, 3H, 21-CH₃, J=5.0), 2.52 (m, 2H, CH₂), 3.385 (q, 2H, CH₂,J=5.0), 3.60 (m, 1H, 3-CH), 3.97 (s, 1H, 12-CH), 6.72 (broad s, 1H, NH)ppm.

¹³C NMR (62.9 MHz) δ=12.7 (CH₃, C18), 17.5 (CH₃, C21), 23.1 (CH₃, C19),23.7 (CH₂), 23.8 (CH₂) 25.2 (CH₂, C15), 26.1 (CH₂) 27.1 (CH₂, C7), 27.5(CH₂, C6), 28.6 (CH₂, C16), 30.5 (CH₂, C2), 31.6 (CH₂, C23), 33.3 (CH₂,C22), 33.6 (CH, C9), 34.1 (C, C10), 35.2 (CH₂, C1), 35.2 (CH, C20), 35.4(CH₂, C4), 36.0 (CH, C8), 36.5 (CH, C5), 42.0 (C, C13), 46.5 (CH, C17),47.0 (CH₂) 48.3 (CH, C14), 54.2 (CH₂), 57.4 (CH₂) 71.6 (CH, C3), 73.0(CH₂, C12), 173.9 (CO, C24) ppm.

IR=3300 (OH), 2925 (alkyl), 2861 (alkyl), 1642 (C═O), 1543, 1437, 1313,1040 (R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 503.4202 (M+H)⁺; calculated for C₃₁H₅₅N₂O₃ 503.4207;1 ppm.

Synthesis of(4R)—N-(4-benzoylphenyl)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanamide(111)

Lithocholic acid (2.0 g 0.005 mol) was dissolved in tetrahydrofuran (60mL) with N-methylmorpholine (1.07 mL, 0.01 mol). The solution was cooledfor 10 minutes in cold water. Ethyl chloroformate (0.54 mL, 0.005 mol)was then added dropwise over a ten minute period. Once added, the coldwater was removed and the solution was stirred for 2 hours. After 2hours amino-benzophenone (1.5 g, 0.007 mol) was added and the solutionwas stirred for a further 48 hours. Water (50 mL) was then added and thesolution was extracted with ethyl acetate (3×50 mL). The organic layerswere combined and washed with saturated sodium hydrogen carbonatesolution (3×50 mL) and 2 M hydrochloric acid solution (3×50 mL). Theorganic layer was dried over magnesium sulphate. The solvent wasevaporated under reduced pressure and the product was triturated withethyl acetate to produce a white powder.

Yield 0.57 g, 0.001 mol, 19%.

Melting point: 228.6-230.8° C.

¹H NMR (DMSO) (250 MHz) δ=0.62 (s, 3H, 18-CH₃) 0.87 (s, 3H, 19-CH₃) 0.92(d, 2H, CH₂ J=7.5) 4.44 (d, 1H, 3-OH, J=5.0) 7.52-7.805 (m, 9H, Ar—CH),10.20 (s, 1H, NH) ppm.

¹³C NMR (62.9 MHz) δ=11.8 (CH₃, C18), 18.3 (CH₃, C21), 20.3 (CH₂, C11),23.2 (CH₃, C19), 23.8 (CH₂, C15), 26.1 (CH₂, C7), 26.8 (CH₂, C6), 27.7(CH₂, C16), 30.3 (CH₂, C2), 31.1 (CH₂, C22), 33.4 (CH₂, C23), 34.1 (C,C10), 34.9 (CH, C20), 35.1 (CH₂, C1), 35.3 (CH, C8), 41.4 (CH, C5), 42.2(C, C13), 55.5 (CH, C17), 56.0 (CH, C14), 69.8 (CH, C3), 118.1, 128.4,129.3, 131.0, 131.1, 132.1, 137.5, 143.5 (Ar—CH) 172.3 (CO, C24), 194.4(CO) ppm.

IR=3488 (NH), 3249 (OH), 2925 (alkyl), 2857 (alkyl), 1675 (C═O), 1586,1296, 1245, 1168 (C═O ester stretch), 1031 (R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 556.3781 (M+H)⁺; calculated for C₃₇H₅₀NO₃ 556.3785;0.8 ppm.

Synthesis of(4R)—N-(4-benzoylphenyl)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanamide(112)

Deoxycholic acid (2.0 g 0.005 mol) was dissolved in 1, 4 dioxane (60 mL)with N-methylmorpholine (1.07 mL 0.01 mol). The solution was cooled for10 minutes in cold water. Ethyl chloroformate (0.69 mL 0.006 mol) wasthen added dropwise over a ten minute period. Once added, the cold waterwas removed and the solution was stirred for 2 hours. After 2 hoursamino-benzophenone (1.5 g 0.007 mol) was added and the solution wasstirred for a further 24 hours. Water (50 mL) was then added and thesolution extracted with ethyl acetate (3×50 mL). The organic layers werecombined and washed with saturated sodium hydrogen carbonate solution(3×50 mL) and 2 M hydrochloric acid (3×50 mL). The organic layer wasdried over magnesium sulphate. Solvent was evaporated under reducedpressure and triturated with ethyl acetate to produce a white powder.

Yield; 0.24 g, 0.0004 mol, 8%.

Melting point: 220-227.6° C.

¹H NMR (DMSO) (250 MHz) δ=0.60 (s, 3H, 18-CH₃), 0.85 (s, 3H, 19-CH₃),0.97 (d, 3H, 21-CH₃, J=5.0), 3.80 (s, 1H, 3-CH), 4.03 (s, 1H, 12-CH),4.21 (d, 1H, 3-OH, J=2.5), 4.46 (d, 1H, 12-OH, J=5.0), 7.52-7.79(multiple overlapping multiplets, 9H, Ar—CH), 10.26 (s, 1H, NH) ppm.

¹³C NMR (62.9 MHz) δ=12.4 (CH₃, C18), 17.0 (CH₃, C21), 23.0 (CH₃, C19),26.9 (CH₂, C7), 27.2, (CH₂, C2), 32.9 (CH₂, C22), 33.5 (CH, C9), 33.7(C, C10), 35.0 (CH₂, C1), 35.6 (CH, C20), 45.9 (C, C13), 46.1 (CH, C17),47.2 (CH, C14), 70.9 (CH, C3), 118.1 (CH), 128.4 (CH), 129.3 (CH), 131.1(CH), 132.1 (CH), 143.5 (CH), 172.4 (CO, C24), 194.5 (CO) ppm.

IR=3462 (OH), 2921 (alkyl), 2861 (alkyl), 1675 (C═O), 1586 (C═O), 1441,1279, 1168.78 (C═O ester stretch), 1036 (R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 572.3730 (M+H)⁺; calculated for C₃₇H₅₀NO₄ 572.3734;0.8 ppm.

Synthesis of(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-1-[4-(4-pyridyl)piperazin-1-yl]pentan-1-one(113)

Lithocholic acid (0.5 g, 0.001 mol) was dissolved in tetrahydrofuran (15mL) with triethylamine (0.20 mL, 0.001 mol). The solution was cooled for10 minutes in cold water. Ethyl chloroformate (0.2 mL, 0.001 mol) wasthen added dropwise over a ten minute period. Once added, the cold waterwas removed and the solution was stirred for 2 hours. After 2 hours1-(4-pyridyl)piperazine (0.21 mL, 0.001 mol) was added and the solutionwas stirred for 24 hours. Water (50 mL) was then added and the solutionextracted with ethyl acetate (3×50 mL). The organic layers were combinedand washed with saturated sodium hydrogen carbonate solution (3×50 mL).Due to impurities, re-dissolved in a methanol/water solution (20 mL50/50) and extracted with chloroform (4×20 mL). The combined organiclayers were dried over magnesium sulphate. The solvent was evaporatedunder reduced pressure to produce a white powder.

Yield; 0.03 g, 0.00005 mol, 4%.

Melting point: 150-157° C.

¹H NMR (CDCl₃) (250 MHz) δ=0.65 (s, 3H, 18-CH₃), 0.92 (s, 3H, 19-CH₃),3.35 (m, 4H, CH₂), 3.64 (m, 2H, CH₂), 6.68 (d, 2H, Ar—CH, J=5.0), 8.31(s, 2H, Ar—CH) ppm.

¹³C NMR (62.9 MHz) δ=12.0 (CH₃, C18), 18.6 (CH₃, C21), 20.8 (CH₂, C11),23.3 (CH₃, C19), 24.3 (CH₂, C15), 26.3 (CH₂, C7), 27.1 (CH₂, C6), 28.3(CH₂, C16), 30.2 (CH₂, C2), 31.3 (CH₂, C22), 34.5 (C, C10), 35.4 (CH,C20), 35.8 (CH₂, C1), 36.4 (CH₂, C4), 40.1 (CH₂, C12), 40.4 (CH, C9),40.7 (CH, C5), 42.1 (C, C13), 42.7 (CH₂), 44.7 (CH₂), 45.8 (CH₂), 45.9(CH₂) 55.9 (CH, C17), 56.5 (CH, C14), 71.8 (CH, C3), 108.5 (CH), 129.7(CH), 149.9 (CH), 150.0 (CH), 154.6 (CH), 172.7 (CO, C24) ppm.

IR=3387 (OH), 2925 (alkyl), 2852 (alkyl), 1641 (C═O), 1445, 1236, 1044,989 (R₂CH—OH) cm⁻¹.

MS (ES) m/z=Found 522.4048 (M+H)⁺; calculated for C₃₁H₅₇N₂O₄ 521.4313;1.5 ppm

Synthesis of(4R)—N-(4-aminobutyl)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanamide(114)

Methyl deoxycholate (1.0 g 0.009 mol) was dissolved in methanol (10 mL).1,4 Diaminobutane (2.47 mL 0.02 mol) was added and the solution wasstirred for a further 72 hours. Water (50 mL) was then added and theresultant precipitate was collected by vacuum filtration, washed withwater (3×20 mL) and dried under vacuum to produce a white powder.

Yield; 0.29 g, 0.0006 mol, 25%.

Melting point: 137.4-141° C.

¹H NMR (DMSO) (250 MHz) δ=0.58 (s, 3H, 18-CH₃), 0.84 (s, 3H, 19-CH₃),0.91 (d, 3H, 21-CH₃, J=5.0), 2.99 (q, 2H, CH₂, J=7.5), 3.6 (m, 1H,3-CH), 3.78 (s, 1H, 12-CH), 7.73 (broad s, 1H, NH) ppm.

¹³C NMR (62.9 MHz) δ=12.4 (CH₃, C18), 17.0 (CH₃, C21), 23.0 (CH₃, C19),23.4 (CH₂, C15), 26.0, (CH₂), 26.6 (CH₂), 26.9 (CH₂) 27.1 (CH₂, C7),28.5 (CH₂, C16), 30.2 (CH₂, C11), 30.5 (CH₂, C2), 31.7 (CH₂, C23), 32.5(CH₂, C22), 32.8 (CH, C9), 33.7 (C, C10), 35.0 (CH₂, C1), 35.1 (CH,C20), 35.6 (CH₂, C4), 36.2 (CH, C8), 41.3 (CH, C5), 41.5 (CH) 45.9 (C,C13), 46.1 (CH, C17), 47.4 (CH, C14), 69.8 (CH, C3), 69.8 (CH₂, C12),172.2 (CO, C24)v ppm.

IR=3322 (OH), 2929 (alkyl), 2857 (alkyl), 1624 (C═O), 1539, 1437, 1360,1040 (R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 463.3886 (M+H)⁺; calculated for C₂₈H₅₁N₂O₃ 463.3894;1.8 ppm.

Synthesis of(4R)—N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanamide(115)

Lithocholic acid (2.0 g, 0.005 mol) was dissolved in toluene (40 mL). 2,2′-(ethylenedioxy)bis(ethylamine) (7.8 mL, 0.052 mol) was added and thesolution was heated at reflux for 5 days. Water (50 mL) was then addedand resultant precipitate collected by vacuum filtration, with furtherwashes of water (3×20 mL). The product was dried under vacuum to producean off-white powder.

Yield, 1.62 g, 0.003 mol, 60.22%

Melting point: 81.1-85.5° C.

¹H NMR (CDCl₃) (250 MHz) δ=0.64 (s, 3H, 18-CH₃), 0.92 (s, 3H, 19-CH₃),2.91 (broad s, 2H, CH₂), 3.46-3.63 (multiple overlapping multiplets,10H, CH₂), 6.24 (broad s, 1H, NH) ppm.

¹³C NMR (62.9 MHz) δ=12.0 (CH₃, C18), 18.4 (CH₃, C21), 19.9 (CH₂) 20.8(CH₂, C11), 23.3 (CH₃, C19), 24.2 (CH₂, C15), 26.4 (CH₂, C7), 27.2 (CH₂,C6), 28.2 (CH₂, C16), 30.5 (CH₂, C2), 31.7 (CH₂, C22), 33.4 (CH₂, C23),34.5 (C, C10), 35.3 (CH, C20), 35.5 (CH₂, C1), 35.8 (CH, C8), 36.4 (CH₂,C4), 39.0 (CH₂, C12), 39.1 (CH, C9), 40.2 (CH₂), 40.4 (CH₂) 42.1 (CH,C5), 42.7 (C, C13), 56.0 (CH, C17), 56.5 (CH, C14), 70.1 (CH₂), 70.5(CH, C3), 71.7 (CH₂), 173.7 (CO, C24) ppm.

IR=3356 (OH), 2921 (alkyl), 2857 (alkyl), 1641 (C═O), 1552, 1441, 1300,1104 (C═O ester stretch), 1053 (R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 507.4149 (M+H)⁺; calculated for C₃₀H₅₅N₂O₄ 507.4156;1.4 ppm.

Synthesis of(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-N-[2-[2-[2-(2methylprop-2-enoylamino)ethoxy]ethoxy]ethyl]pentanamide (116)

Compound 115 (0.5 g 0.0009 mol) was dissolved in anhydroustetrahydrofuran (10 mL) with triethylamine (0.20 mL, 0.001 mol).Methacrylic anhydride (0.30 mL, 0.0018) was then added. The solution wasstirred for 48 hours, protected from sunlight by tin foil at ambientroom temperature. Water (30 mL) was then added and the resultingprecipitate was collected by vacuum filtration, washed with water (3×20mL) and dried under vacuum.

Yield; 0.07 g, 0.0001 mol, 12.5%.

Melting point: 57.3-59.0° C.

¹H NMR (DMSO) (250 MHz) δ=0.60 (s, 3H, 18-CH₃), 0.87 (s, 3H, 19-CH₃),1.84 (s, 3H, acryloyl-CH₃), 3.154-3.50 (m, 13H, CH/CH₂), 4.42 (d, 1H,3-OH, J=5.0), 5.31 (s, 1H, ═CH), 5.64 (s, 1H, ═CH), 7.78 (broad s, 1H,NH), 7.91 (broad s, 1H, NH) ppm.

¹³C NMR (62.9 MHz) δ=11.8 (CH₃, C18), 18.2 (CH₃, C21), 18.6 (CH), 20.3(CH₂, C11), 23.2 (CH₃, C19), 23.8 (CH₂, C15), 26.1 (CH₂, C7), 26.8 (CH₂,C6), 27.7 (CH₂, C16), 30.3 (CH₂, C2), 31.5 (CH₂, C22), 32.2 (CH₂, C23),34.1 (C, C10), 34.9 (CH, C20), 35.1 (CH₂, C1), 35.3 (CH, C8), 41.4 (CH,C5), 42.2 (C, C13), 55.2 (CH, C17), 56.0 (CH, C14), 68.8 (CH₂), 69.1(CH₂), 69.5 (CH₂), 69.8 (CH, C3), 118.9 (CH₂), 139.8 (CH₂), 167.4 (CO)172.5 (CO, C24) ppm.

IR=3415 (NH), 3292 (OH), 2933 (alkyl), 2861 (alkyl), 1658 (C═O), 1590(C═O), 1394, 1249 (C═O ester stretch), 1036 (R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 575.4412 (M+H) ⁺; calculated for C₃₄H₅₉N₂O₅575.4418; 1.1 ppm.

Synthesis of(4R)—N-(4-acetylphenyl)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanamide(117)

Deoxycholic acid (2.0 g 0.005 mol) was dissolved in 1, 4 dioxane (60 mL)with triethylamine (1.32 mL, 0.01 mol). The solution was cooled for 10minutes in cold water. Ethyl chloroformate (0.40 mL, 0.003 mol) was thenadded dropwise over a ten minute period. Once added, the cold water wasremoved and the solution was stirred for 2 hours. After 2 hours4′-aminoacetophenone (0.75 g, 0.005 mol) was added and the solution wasstirred for a further 48 hours. Water (50 mL) was then added and theresulting precipitate was collected by vacuum filtration. The crudeproduct was triturated with ethyl acetate to produce a white powder.

Yield; 0.65 g, 0.001 mol, 25%.

¹H NMR (DMSO) (250 MHz) δ=0.62 (s, 3H, 18-CH₃), 0.86 (s, 3H, 19-CH₃),2.52 (s, 3H, ketone-CH₃), 3.81 (broad s, 1H, 12-CH), 4.21 (d, 1H, 3-OH,J=2.5), 4.46 (d, 1H, 12-OH, J=2.5), 7.73 (d, 2H, Ar—CH, J=10.0), 7.92(d, 2H, Ar—CH, J=7.5), 10.21 (s, 1H, NH) ppm.

IR=3475 (OH), 2921 (alkyl), 2861 (alkyl), 1684 (C═O), 1646 (C═O), 1594,1539, 1407, 1044 (C═O ester stretch) cm⁻¹.

MS (+ESI) m/z=Found 510.3570 (M+H)⁺; calculated for C₃₂H₄₈NO₄ 510.3578;1.3 ppm.

Synthesis of ethane;(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-1-[4-(2-hydroxyethyl)piperazin-1-yl]pentan-1-one(118)

Lithocholic acid (4.0 g 0.01 mol) was dissolved in tetrahydrofuran (120mL) with triethylamine (1.30 mL, 0.01 mol). The solution was cooled for10 minutes in cold water. Ethyl chloroformate (1.02 mL, 0.009 mol) wasthen added dropwise over a ten minute period. Once added, the cold waterwas removed and the solution was stirred for 2 hours. After 2 hours1-(2-hydroxyethylpiperazine) (1.5 mL, 0.01 mol) was added and thesolution was stirred for a further 48 hours. Water (50 mL) was thenadded and the solution was extracted with ethyl acetate (3×50 mL). Theorganic layers were combined and washed with saturated sodium hydrogencarbonate solution (3×50 mL). The organic layer was dried over magnesiumsulphate. The solvent was evaporated under reduced pressure and theproduct was recrystallized from ethyl acetate to produce a white powder.

Yield; 2.27 g, 0.004 mol, 43%.

Melting point: 153.0-154.8° C.

¹H NMR (CDCl₃) (250 MHz) δ=0.65 (s, 3H, 18CH₃), 0.92 (s, 3H, 19-CH₃),2.50 (m, 4H, CH₂), 2.58 (t, 2H, CH₂, J=5.0), 3.49 (t, 2H, CH₂, J=2.5),3.65 (t, 4H, CH₂, J=5.0) ppm.

¹³C NMR (62.9 MHz) δ=12.0 (CH₃, C18), 18.5 (CH₃, C21), 20.8 (CH₂, C11),23.3 (CH₃, C19), 24.2 (CH₂, C15), 26.4 (CH₂, C7), 27.2 (CH₂, C6), 28.2(CH₂, C16), 30.2 (CH₂, C2), 30.5 (CH₂, C22), 31.4 (CH₂, C23), 34.5 (C,C10), 35.3 (CH, C20), 35.6 (CH₂, C1), 35.8 (CH, C8), 36.4 (CH₂, C4),40.2 (CH₂, C12), 40.4 (CH, C9), 41.4 (CH, C5), 42.1 (C, C13), 42.7(CH₂), 45.6 (CH₂), 52.6 (CH₂), 53.1 (CH₂) 56.0 (CH, C17), 56.5 (CH,C14), 57.7 (CH₂), 59.3 (CH₂) 71.8 (CH, C3), 172.1 (CO, C24) ppm.

IR=3381 (OH), 2916 (alkyl), 2840 (alkyl), 1620 (C═O), 1445, 1258, 1044(R₂CH—OH) cm⁻¹.

MS (ES) m/z=Found 489.4045 (M+H)⁺; calculated for C₃₀H₅₃N₂O₃ 489.4051;1.2 ppm.

Synthesis of(4R)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-1-[4-(2-hydroxyethyl)piperazin-1-yl]pentan-1-one(119)

Deoxycholic acid (4.0 g 0.01 mol) was dissolved in 1, 4 dioxane (120 mL)with triethylamine (2.70 mL 0.02 mol). The solution was cooled for 10minutes in cold water. Ethyl chloroformate (0.8 mL 0.005 mol) was thenadded dropwise over a ten minute period. Once added, the cold water wasremoved and the solution was stirred for 2 hours. After 2 hours1-(2-hydroxyethylpiperazine) (1.32 mL 0.01 mol) was added and thesolution was stirred for 48 hours. Water (50 mL) was then added and thesolution extracted with ethyl acetate (3×50 mL). The organic layers werecombined and washed with saturated sodium hydrogen carbonate solution(3×50 mL). The organic layer was dried over magnesium sulphate. Solventwas evaporated under reduced pressure and recrystallized from methanolto produce a white powder.

Yield; 2.41 g, 0.004 mol, 46.8%.

Melting point: 241.0-243.9° C.

¹H NMR (CDCl₃/MeOH) (250 MHz) δ=0.70 (s, 3H, 18-CH₃), 0.93 (s, 3H,19-CH₃), 2.58 (multiple overlapping multiplets, 6H, CH₂), 3.53 (broad t,3H, 3-CH/CH₂), 3.65-3.72 (multiple overlapping multiplets, 4H, CH₂),3.96 (broad s, 1H, 12-CH) ppm.

IR=3407 (OH), 2929 (alkyl), 1620 (C═O), 1454, 1215, 1044 (R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 505.3994 (M+H)⁺; calculated for C₃₁H₅₇N₂O₄ 505.4000;1.2 ppm.

Synthesis of(4R)—N-[3-(dibutylamino)propyl]-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanamide(120)

Lithocholic acid (0.5 g 0.001 mol) was dissolved in tetrahydrofuran (15mL) with triethylamine (0.20 mL 0.001 mol). The solution was cooled for10 minutes in cold water. Ethyl chloroformate (0.2 mL 0.001 mol) wasthen added dropwise over a ten minute period. Once added, the cold waterwas removed and the solution was stirred for 2 hours. After 2 hours3-(dibutylamino)-1-propylamine (0.25 mL, 0.001 mol) was added and thesolution was stirred for 24 hours. Water (50 mL) was then added and thesolution extracted with ethyl acetate (3×50 mL). The organic layers werecombined and washed with saturated sodium hydrogen carbonate solution(3×50 mL). This crude material was further purified using columnchromatography (8:2 ethyl acetate/methanol). The compound containingfractions were dried over magnesium sulphate. Solvent was evaporatedunder reduced pressure to produce a white powder.

Yield; 0.24 g, 0.0004 mol, 33%.

Melting point: 62.9-69.3° C.

¹H NMR (CDCl₃) (250 MHz) δ=0.64 (s, 3H, 18-CH₃), 0.93 (s, 3H, 19-CH₃),0.94 (m, 6H, CH₃), 1.35 (m, 8H, CH₂), 2.45 (t, 4H, CH₂, J=5.0), 3.32 (q,2H, CH₂, J=5.0), 7.51 (broad s, 1H, NH) ppm.

¹³C NMR (62.9 MHz) δ=12.0 (CH₃, C18), 14.0 (CH), 18.3 (CH₃, C21), 20.7(CH₂, C11), 23.3 (CH₃, C19), 24.2 (CH₂, C15), 25.1 (CH₂), 26.4 (CH₂,C7), 27.2 (CH₂, C6), 28.2 (CH₂, C16), 28.4 (CH₂), 28.5 (CH₂) 30.4 (CH₂,C2), 30.5 (CH₂, C22), 33.8 (CH₂, C23), 34.5 (C, C10), 35.3 (CH, C20),36.4 (CH₂, C1), 39.4 (CH₂, C4), 40.1 (CH₂, C12), 40.4 (CH, C9), 42.0(CH, C5), 42.1 (C, C13), 42.7 (CH₂) 56.1 (CH, C17), 56.4 (CH, C14), 71.6(CH, C3), 173.6 (CO, C24) ppm.

IR=3296 (OH), 2916 (alkyl), 2861 (alkyl), 1650 (C═O), 1548, 1441, 1377,1070, 1066, 1036 (R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 545.5032 (M+H)⁺; calculated for C₃₅H₆₅N₂O₂ 545.5041;1.6 ppm.

Synthesis of(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-N-octadecyl-pentanamide(121)

Lithocholic acid (2.0 g 0.005 mol) was dissolved in tetrahydrofuran (60mL) with 4-methylmorpholine (2.14 mL, 0.02 mol). The solution was cooledfor 10 minutes in cold water. Ethyl chloroformate (0.51 mL, 0.005 mol)was then added dropwise over a ten minute period. Once added, the coldwater was removed and the solution was stirred for 2 hours. After 2octadecylamine (1.43 mL, 0.005 mol) was added and the solution wasstirred for a further 21 hours. Water (50 mL) was then added and thesolution was extracted with ethyl acetate (3×50 mL). The crude productwas purified by column chromatography (100% ethyl acetate). The productcontaining fractions were dried over magnesium sulphate. The solvent wasevaporated under reduced pressure to produce a white powder.

Yield; 0.8 g, 0.001 mol, 23.9%.

Melting point: 94.8-95.4° C.

¹H NMR (250 MHz) CDCl₃ δ=0.64 (s, 3H, 18-CH₃), 0.92 (s, 3H, 19-CH₃),1.25 (s, 30H, aliphatic CH₂) 3.23 (q, 3H, chain terminal CH₃, J=5.0),3.64 (m, 1H, 3-CH), 5.36 (broad s, 1H, NH) ppm.

¹³C NMR (62.9 MHz) δ=12.0 (CH₃, C18), 18.4 (CH₃, C21), 20.8 (CH₂, C11),22.7 (CH₃, C19), 23.3 (CH₂, C15), 24.2 (CH₂) 26.4 (CH₂, C7), 26.9 (CH₂,C6), 27.2 (CH₂, C16), 28.2 (CH₂), 29.3 (CH₂), 29.5 (CH₂), 29.7 (CH₂),30.5 (CH₂, C2), 31.8 (CH₂, C22), 31.9 (CH₂, C23), 33.7 (C, C10), 35.3(CH, C20), 35.4 (CH₂, C1), 35.8 (CH, C8), 39.5 (CH₂, C4), 40.2 (CH₂,C12), 40.4 (CH, C9), 42.1 (C, C5), 56.0 (CH, C17), 56.5 (CH, C14), 71.9(CH, C3), 185.3 (CO, C24) ppm.

IR=3411 (NH), 3325 (OH), 2912 (alkyl), 2849 (alkyl), 1653 (C═O), 1544,1464, 1367, 1308, 1040 (R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 628.6205 (M+H)⁺; calculated for C₄₂H₇₈NO₂ 628.6207;0.6 ppm.

Synthesis of(4R)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-N-octadecyl-pentanamide(122)

Deoxycholic acid (2.0 g 0.005 mol) was dissolved in 1, 4 dioxane (60 mL)with triethylamine (1.32 mL, 0.01 mol). The solution was cooled for 10minutes in cold water. Ethyl chloroformate (0.40 mL, 0.003 mol) was thenadded dropwise over a ten minute period. Once added, the cold water wasremoved and the solution was stirred for 2 hours. After 2 hoursoctadecylamine (1.37 g, 0.005 mol) was added and the solution wasstirred for a further 48 hours. Water (50 mL) was then added and thesolution was extracted with ethyl acetate (3×50 mL). The organic layerswere combined and washed with saturated sodium hydrogen carbonatesolution (3×50 mL). The organic layer was dried over magnesium sulphate.The solvent was evaporated under reduced pressure and the product wasrecrystallized from ethyl acetate to produce a transparent glass-likesolid.

Yield; 2.32 g, 0.003 mol, 70%.

Melting point: 54.2-57.3° C.

¹H NMR (CDCl₃) (250 MHz) δ=0.68 (s, 3H, 18-CH₃), 0.91 (s, 3H, 19-CH₃),1.25 (s, 32H, aliphatic CH₂) 3.22 (q, 2H, CH₂, J=7.5), 3.61 (m, 1H,3-CH), 3.98 (1H, 12-CH), 5.53 (broad s, 1H, NH) ppm.

¹³C NMR (62.9 MHz) δ=12.7 (CH₃, C18), 14.1 (CH₂), 17.4 (CH₃, C21), 22.6(CH₂), 23.1 (CH₃, C19), 23.6 (CH₂, C15), 26.1 (CH), 26.9 (CH₂, C7), 28.6(CH₂, C16), 29.3 (CH₂, C11), 29.5 (CH₂, C2), 29.7 (CH₂), 30.5 (CH) 31.7(CH₂, C23), 31.9 (CH₂, C22), 33.5 (CH, C9), 33.6 (CH), 34.1 (CH), 35.2(C, C10), 35.2 (CH) 36.0 (CH₂, C1), 36.4 (CH, C20), 39.5 (CH₂, C4), 42.1(CH, C5), 46.5 (C, C13), 47.2 (CH, C17), 48.2 (CH, C14), 71.7 (CH, C3),73.1 (CH₂, C12), 173.4 (CO, C24) ppm.

IR=3292 (OH), 2912, 2852 (alkyl), 1637 (C═O), 1548, 1189.78 (C═O esterstretch), 1036 (RCHOH) cm⁻¹.

MS (+ESI) m/z=Found 644.5971 (M+H)⁺; calculated for C₄₂H₇₈NO₃ 644.5976;0.8 ppm.

Synthesis of2-[[(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]ethyl-trimethyl-ammoniumiodide (123)

Compound 101 (0.2 g 0.0004 mol) was dissolved in chloroform (10 mL) withIodomethane (0.31 mL 0.002 mol). The solution was left overnight atwhich point a precipitate had formed which was collected by vacuumfiltration, washed with water (3×20 mL) and dried under vacuum toproduce a white powder.

Yield; 0.212 g, 0.0003 mol, 81.5%.

Melting point: 212.8-216.1° C.

¹H NMR (MeOD) (250 MHz) δ=0.69, (s, 3H, 18-CH₃), 0.94 (s, 3H, 19-CH₃),3.19, (s, 9H, 3×CH₃), 3.45 (q, 2H, CH₂, J=7.5), 3.55 (m, 1H, 3-CH), 3.64(t, 2H, CH₂, J=7.5) ppm.

¹³C NMR (62.9 MHz) δ=11.0 (CH₃), 12.5 (CH₃), 17.3 (CH₃), 18.8 (CH₂),21.9 (CH) 23.9 (CH₂), 27.6 (CH), 28.3 (CH), 31.2 (CH), 33.0 (CH), 33.9(CH), 36.9 (CH₂), 37.2 (CH₂), 41.9 (CH), 43.5 (C, C13), 57.4 (CH, C17),57.9 (CH, C14), 65.8 (CH₂) 72.4 (CH), 181.2 (CO) ppm.

IR=3368 (NH), 3245 (OH), 2938 (alkyl), 2852 (alkyl) 1639 (C═O), 1560,1441, 1258 (C═O ester stretch), 1040 (R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 461.4105 M⁺; calculated for C₂₉H₅₃N₂O₂ 461.4102; 0.7ppm.

Synthesis of2-[[(4R)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]ethyl-trimethyl-ammoniumiodide (124)

Compound 102 (0.15 g 0.0003 mol) was dissolved in chloroform (5 mL).Iodomethane (0.22 mL, 0.001 mol) was added and the solution was stirredovernight at ambient temperature. The resultant precipitate wascollected via vacuum filtration and dried in a desiccator overnight togive the product as an off-white solid.

Yield 0.09 g, 0.0001 mol, 78.9%.

Melting point: 160.1-169.1° C.

¹H NMR (MeOD) (250 MHz) δ=0.70 (s, 3H, 18-CH₃), 0.93 (s, 3H, 19-CH₃),1.02 (d, 3H, 21-CH₃, J=5.0), 3.20 (s, 9H, 3×CH₃), 3.47 (t, 2H, CH₂,J=7.5), 3.64 (t, 2H, CH₂, J=5.0), 3.95 (s, 1H, 12-CH) ppm.

¹³C NMR (62.9 MHz) δ=13.2 (CH₃, C18), 17.6 (CH₃, C21), 23.7 (CH₃, C19),24.8 (CH₂, C15), 27.4 (CH₂, C7), 28.4 (CH₂, C6), 28.7 (CH₂, C16), 29.9(CH₂, C11), 31.0 (CH₂, C2), 33.0 (CH₂, C23), 33.8 (CH₂), 34.6 (CH₂),36.4 (CH₂, C1), 36.8 (CH, C20), 37.2 (CH₂, C4), 37.4 (CH, C8), 43.6 (CH,C5), 54.0 (CH), 54.0 (CH), 54.1 (CH), 72.5 (CH, C3), 74.0 (CH₂, C12),177.3 (CO, C24) ppm.

IR=3377 (NH), 3249 (OH), 2921 (alkyl), 2861 (alkyl), 1641 (C═O), 1573,1454, 1373, 1249, 1031 (R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 477.4060 M⁺; calculated for C₂₉H₅₃N₂O₃ 477.4051; 1.9ppm.

Synthesis ofethyl-[3-[[(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]propyl]-dimethyl-ammoniumiodide (125)

Compound 101 (0.2 g 0.0004 mol) was dissolved in chloroform (10 mL).Ethyl Iodide (0.34 mL, 0.001 mol) was added and the solution was stirredovernight at ambient temperature. The resulting precipitate wascollected by vacuum filtration and was dried overnight in a desiccatorto give the product as an off-white solid.

Yield; 0.2 g, 0.0003 mol, 76%.

Melting point: 120.0-123.0° C.

¹H NMR (MeOD) (250 MHz) δ=0.69 (s, 3H, 18-CH₃), 0.95 (s, 3H, 19-CH₃),3.13 (s, 6H, 2×CH₃), 3.43 (m, 4H, 3-CH/CH₂), 3.60 (q, 2H, CH₂, J=7.5)ppm.

¹³C NMR (62.9 MHz) δ=8.4 (CH) 12.5 (CH₃, C18), 18.8 (CH₃, C21), 21.4(CH₂, C11), 21.9 (CH₃, C19), 23.9 (CH₂, C15), 25.2 (CH₂, C7), 28.3 (CH₂,C16), 29.3 (CH₂, C2), 31.2 (CH₂, C23), 33.0 (CH₂), 33.9 (CH₂), 34.2 (C,C10), 35.6 (CH, C20), 36.4 (CH₂, C1), 37.1 (CH, C8), 37.2 (CH₂, C4),41.5 (CH, C9), 41.9 (CH, C5), 43.5 (C, C13), 43.9 (CH₂), 51.1 (CH₂) 57.3(CH, C17), 57.9 (CH, C14), 62.4 (CH) (CH, C3), 177.3 (CO, C24) ppm.

IR=3373 (OH), 2942 (alkyl), 2844 (alkyl), 1646 (C═O), 1441, 1270, 1036(R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 475.4247 M⁺; calculated for C₃₀H₅₅N₂O₂ 475.4258; 2.3ppm.

Synthesis of3-[[(4R)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]propyl-ethyl-dimethyl-ammoniumiodide (126)

Compound 102 (0.2 g 0.0004 mol) was dissolved in chloroform (5 mL).Ethyl Iodide (0.62 mL, 0.003 mol) was added and the solution was stirredovernight at ambient temperature. The resulting precipitate wascollected by vacuum filtration and was dried overnight in a desiccator.The product was an off-white solid.

Yield; 0.13 g, 0.0002 mol, 52%.

Melting point: 115.3-120.3° C.

¹H NMR (CDCl₃) (250 MHz) δ=0.70 (s, 3H, 18-CH₃), 0.93 (s, 3H, 19-CH₃),1.02 (d, 2H, CH₂), 2.21 (m, 6H, 3-CH/CH₂), 3.4 (broad s, 2H, CH₂),3.45-3.61 (multiple overlapping multiplets, 8H, CH₂) ppm.

¹³C NMR (62.9 MHz) δ=8.4 (CH₂) 12.4 (CH₃, C18), 16.9 (CH₃, C21), 21.9(CH₂) 22.9 (CH₃, C19), 24.0 (CH₂, C15), 26.6 (CH₂, C7), 27.9 (CH₂, C16),29.1 (CH₂, C11), 30.3 (CH₂, C2), 32.2 (CH₂, C23), 33.1 (CH₂, C22), 34.0(CH, C9), 34.5 (C, C10), 35.6 (CH₂, C1), 36.0 (CH, C20), 36.4 (CH₂, C4),36.6 (CH, C8), 42.8 (CH, C5), 55.6 (CH), 57.1 (CH), 63.2 (CH) 71.7 (CH,C3), 73.2 (CH₂, C12), 176.6 (CO, C24) ppm.

IR=3368 (NH), 3253 (OH), 2921 (alkyl), 2852 (alkyl), 1650 (C═O), 1522,1445, 1364, 1253, 1036 (R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 517.4354 M⁺; calculated for C₃₂H₅₇N₂O₃ 517.4364; 1.9ppm.

Synthesis ofallyl-[2-[[(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]ethyl]-dimethyl-ammoniumbromide (127)

Compound 101 (0.2 g, 0.0004 mol) was dissolved in a solution ofchloroform (10 mL). Allyl bromide (0.27 mL, 0.002 mol) was added and thesolution was stirred overnight at which point a precipitate was formed.The precipitate was collected by vacuum filtration and was driedovernight in a desiccator. The product was an off-white solid.

Yield; 0.13 g, 0.0002 mol, 54%.

Melting point: 198.7-203.8° C.

¹H NMR (MeOD) (250 MHz) δ=0.68 (s, 3H, 18-CH₃), 0.94 (s, 3H, 19-CH₃),3.12 (s, 6H, 2×CH₃), 3.39 (t, 2H, CH₂), 3.65 (t, 2H, CH₂), 5.70 (m, 2H,CH₂), 6.10 (m, 1H, ═CH—) ppm.

¹³C NMR (62.9 MHz) δ=12.5 (CH₃, C18), 18.8 (CH₃, C21), 21.9 (CH₂, C11),23.9 (CH₃, C19), 25.2 (CH₂, C15), 27.6 (CH₂, C7), 28.3 (CH₂), 28.0 (CH₂,C16), 33.0 (CH₂) 34.2 (C, C10), 37.1 (CH₂), 41.5 (CH₂), 41.9 (CH₂), 43.8(CH), 57.3 (CH₂), 57.9 (CH₂), 63.0 (CH₂), 72.4 (CH, C3), 126.0 (CH),129.8 (CH), 177.3 (CO, C24) ppm.

IR=3266 (OH), 2929 (alkyl), 2848 (alkyl), 1646 (C═O), 1569, 1420, 1066,1036 (R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 487.4252 M⁺; calculated for C₃₁H₅₅N₂O₂ 487.4258; 1.2ppm.

Synthesis ofallyl-[2-[[(4R)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]ethyl]-dimethyl-ammoniumiodide (128)

Compound 102 (0.15 g, 0.0003 mol) was dissolved in chloroform (5 mL).Allyl bromide (0.20 mL, 0.0016 mol) was added and the solution wasstirred overnight at ambient temperature. The resulting precipitate wascollected by vacuum filtration and was dried overnight in a desiccator.The product was an off-white solid.

Yield; 0.19 g, 0.0001 mol, 53%.

Melting point: 184.2-186.7° C.

¹H NMR (MeOD) (250 MHz) δ=0.70 (s, 3H, CH₃), 0.93 (s, 3H, CH₃), 3.13 (s,6H, CH₃), 3.40 (t, 2H, CH₂, J=7.5), 3.65 (t, 2H, CH₂, J=5.0), 3.95 (s,1H, 12-CH), 4.05 (d, 2H, CH₂, J=5.0), 5.75 (m, 2H, CH₂), 6.10 (m, 1H,═CH—) ppm.

¹³C NMR (62.9 MHz) δ=13.2 (CH₃, C18), 17.6 (CH₃, C21), 23.7 (CH₃, C19),24.8 (CH₂, C15), 27.4 (CH₂, C7), 28.4 (CH₂, C6), 28.7 (CH₂, C16), 29.9(CH₂, C11), 31.1 (CH₂, C2), 33.0 (CH₂, C23), 33.8 (CH₂, C22), 34.8 (CH,C9), 36.9 (CH), 37.2 (CH), 37.4 (CH), 43.6 (CH, C5), 47.5 (CH, C17),48.0 (CH, C14), 63.0 (CH₂), 63.1 (CH₂), 67.9 (CH₂), 68.0 (CH₂), 68.0(CH₂) 72.5 (CH, C3), 74.0 (CH₂, C12), 126.1 (CH₃), 129.8 (CH₂), 177.4(CO, C24) ppm.

IR=3415 (NH), 3245 (OH), 2925 (alkyl), 2852 (alkyl), 1646 (C═O), 1441,1364, 1292 cm⁻¹.

MS (+ESI) m/z=Found 503.4202 M⁺; calculated for C₃₁H₅₅N₂O₃ 503.4207; 1.0ppm.

Synthesis of2-[[(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]ethyl-dimethyl-[(4-vinylphenyl)methyl]ammoniumchloride (129)

Compound 101 (0.2 g 0.0004 mol) was dissolved in chloroform (10 mL).Vinyl benzyl chloride (0.64 mL, 0.004 mol) was added and the solutionwas stirred overnight at ambient temperature. The resulting precipitatewas collected by vacuum filtration and was dried overnight in adesiccator. The product was an off-white solid.

Yield; 0.07 g, 0.0016 mol, 26.9%.

Melting point: 143.8-148.9° C.

¹H NMR (MeOD) (250 MHz) δ=0.66 (s, 3H, 18-CH₃), 0.94 (s, 3H, 19-CH₃),3.09 (s, 6H, 2×CH₃), 3.41 (t, 2H, CH₂, J=7.5), 3.73 (t, 2H, CH₂, J=5.0),4.56 (s, 2H, CH₂), 5.37 (d, 1H, ═CH—, J=12.5), 5.91 (d, 1H, ═CH, J=20.0)6.80 (dd, 1H, ═CH, J=10.0), 7.57 (dd, 4H, Ar—CH, J=5.0) ppm.

¹³C NMR (62.9 MHz) δ=11.7 (CH₃, C18), 18.0 (CH₃, C21), 21.1 (CH₂, C11),23.1 (CH₃, C19), 24.4 (CH₂, C15), 26.8 (CH₂, C7), 27.5 (CH₂, C6), 28.4(CH₂, C16), 30.3 (CH₂, C2), 33.0 (CH₂), 33.9 (CH₂), 35.7 (CH₂), 36.8(CH), 37.2 (CH), 41.9 (CH), 43.5 (CH), 57.4 (CH, C17), 57.9 (CH, C14),63.5 (CH₂) 71.6 (CH, C3), 115.8 (CH), 126.9 (CH), 127.2 (CH), 133.6(CH), 136.2 (CH), 140.8 (CH), 176.5 (CO, C24) ppm.

IR=3339 (NH), 3215 (OH), 2921 (alkyl), 2857 (alkyl), 1667 (C═O), 1646,1445, 1356, 1070 (R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 563.4558 M⁺; calculated for C₃₇H₅₉N₂O₂ 563.4571; 2.3ppm.

Synthesis3-[[(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]propyl-trimethyl-ammoniumiodide (130)

Compound 103 (0.5 g 0.002 mol) was dissolved in a mixture of chloroform(15 mL) and methanol (4 mL). Iodomethane (3.63 mL, 0.02 mol) was addedand the solution was stirred for four days at ambient temperature. Toinduce precipitation the solution was placed in a acetone/dry icemixture (−78° C.). The resulting precipitate was collected by vacuumfiltration and was dried overnight in a desiccator. The product was anoff-white solid.

Yield; 0.42 g, 0.06 mol, 64.6%.

Melting point: 253.8-255.4° C.

¹H NMR (CDCl₃) (250 MHz) δ=0.69 (s, 3H, 18-CH₃), 0.94 (s, 3H, 19-CH₃),3.13 (s, 9H, 3×CH₃), 3.54 (m, 1H, 3-CH) ppm.

¹³C NMR (62.9 MHz) δ=10.9 (CH₃, C18), 17.3 (CH₃, C21), 20.4 (CH₂, C11),22.4 (CH₂), 23.0 (CH₃, C19), 23.7 (CH₂, C15), 26.1 (CH₂, C7), 26.8 (CH₂,C6), 28.0 (CH₂, C16), 29.6 (CH₂, C2), 31.6 (CH₂, C22), 32.5 (CH₂, C23),34.1 (C, C10), 34.9 (CH, C20), 35.4 (CH₂, C1), 35.6 (CH, C8), 35.7 (CH₂,C4), 40.0 (CH₂, C12), 40.4 (CH, C9), 42.0 (CH, C5), 42.4 (C, C13), 52.2(CH, C17), 55.8 (CH, C14), 56.8 (CH), 64.2 (CH₂) 70.8 (CH, C3), 175.7(CO, C24) ppm.

IR=3386 (OH), 2921 (alkyl), 2852 (alkyl), 1641 (C═O), 1552, 1441, 1368,1253 (C═O ester stretch), 1031 (R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 475.4256 M⁺; calculated for C₃₀H₅₅N₂O₂ 475.4258; 0.4ppm.

Synthesis of3-[[(4R)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]propyl-trimethyl-ammoniumiodide (131)

Compound 104 (0.1 g 0.0002 mol) was dissolved in chloroform (5 mL).Iodomethane (0.29 mL, 0.002 mol) was added and the solution was stirredovernight at ambient temperature. The resulting precipitate wascollected by vacuum filtration and was dried overnight in a desiccator.The product was an off-white solid.

Yield; 0.09 g, 0.0014 mol, 75%.

Melting point: 150.1-153.3° C.

¹H NMR (MeOD) (250 MHz) δ=0.71 (s, 3H, 18-CH₃), 0.93 (s, 3H, 19-CH₃),1.03 (d, 3H, 21-CH₃, J=7.5), 3.15 (s, 6H, 2×CH₃), 3.53 (m, 1H, 3-CH),3.95 (s, 1H, 12-CH) ppm.

¹³C NMR (62.9 MHz) δ=13.2 (CH₃, C18), 17.7 (CH₃, C21), 23.7 (CH₃, C19),27.4 (CH₂, C7), 28.4 (CH₂, C6), 28.7 (CH₂, C16), 29.9 (CH₂, C11), 31.1(CH₂, C2), 33.1 (CH₂, C23), 35.3 (C, C10), 37.2 (CH₂, C4), 43.6 (CH,C5), 72.5 (CH, C3), 74.0 (CH₂, C12), 177.3 (CO, C24) ppm.

IR=3364 (OH), 2933 (alkyl), 2852 (alkyl), 1654 (C═O), 1548, 1437 cm⁻¹.

MS (ES) m/z=Found 491.4195 M⁺; calculated for C₃₀H₅₅N₂O₃ 491.4207; 2.5ppm.

Synthesis ofallyl-[3-[[(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]propyl]-dimethyl-ammoniumbromide (132)

Compound 103 (0.5 g 0.001 mol) was dissolved in dichloromethane (20 mL).Allyl bromide (1.88 mL, 0.01 mol) was added and the solution was stirredfor a further 5 days. The reaction mixture was chilled to −78° C. Aprecipitate formed. This was collected by vacuum filtration.

Yield; 0.45 g, 0.0007 mol, 71%.

Melting point: 191.1-209.9° C.

¹H NMR (CDCl₃) (250 MHz) δ=0.68 (s, 3H, 18-CH₃), 0.94 (s, 3H, 19-CH₃),3.10 (s, 6H, 2×CH₃), 3.27 (q, 2H, CH₂), 3.54 (m, 1H, 3-CH), 4.01 (d, 2H,CH₂, J=7.5), 5.74 (t, 2H, CH₂, J=10.0), 6.08 (m, 1H, NH) ppm.

¹³C NMR (62.9 MHz) δ=11.1 (CH₃, C18), 17.5 (CH₃, C21), 20.5 (CH₂, C11),22.5 (CH₃, C19), 23.8 (CH₂, C15), 26.2 (CH₂, C7), 26.9 (CH₂, C6), 27.0(CH₂, C16), 29.7 (CH₂, C2), 31.7 (CH₂, C22), 32.5 (CH₂, C23), 34.2 (C,C10), 35.0 (CH, C20), 35.4 (CH₂, C1), 35.7 (CH, C8), 35.9 (CH₂, C4),40.0 (CH₂, C12), 40.4 (CH, C9), 42.0 (CH, C5), 42.4 (C, C13), 55.9 (CH,C17), 56.4 (CH, C14), 61.7 (CH₂), 66.1 (CH₂) 70.9 (CH, C3), 124.8 (CH),128.0 (CH₂), 175.8 (CO, C24) ppm.

IR=3411 (NH), 3270 (OH), 2925 (alkyl), 2857 (alkyl), 1646 (C═O), 1543,1437, 1373, 1036 (R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 501.4405 M⁺; calculated for C₃₁H₅₅N₂O₂.

Synthesis ofcyclopentylmethyl-[3-[[(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]propyl]-dimethyl-ammoniumbromide (133)

Compound 103 (0.5 g 0.001 mol) was dissolved in chloroform (20 mL) withbenzyl bromide (1.37 mL, 0.008 mol) and the solution was stirred for 48hours. The solvent was evaporated under reduced pressure, thenre-dissolved in methanol and washed with petroleum ether 60/80 (3×20mL). The solvent was removed under reduced pressure to produce a whitepowder.

Yield; 0.24 g, 0.0003 mol, 35%.

Melting point: 145-147.9° C.

¹H NMR (MeOD) (250 MHz) δ=0.68 (s, 3H, 18-CH₃), 0.94 (s, 3H, 19-CH₃),3.05 (s, 6H, 2×CH₃), 3.54 (m, 1H, 3-CH), 4.56 (s, 2H, CH₂), 7.55 (broads, 5H, Ar—CH) ppm.

¹³C NMR (62.9 MHz) δ=10.9 (CH₃, C18), 17.3 (CH₃, C21), 20.4 (CH₂, C11),22.4 (CH₃, C19), 22.6 (CH₂, C15), 23.7 (CH) 26.1 (CH₂, C7), 26.8 (CH₂,C6), 27.7 (CH₂, C16), 29.6 (CH₂, C2), 31.6 (CH₂, C22), 32.5 (CH₂, C23),34.1 (C, C10), 34.9 (CH, C20), 35.3 (CH₂, C1), 35.6 (CH, C8), 35.6 (CH₂,C4), 39.9 (CH₂, C12), 40.3 (CH, C9), 41.9 (CH, C5), 42.3 (C, C13), 55.8(CH, C17), 56.3 (CH, C14), 61.7 (CH), 67.4 (CH), 70.8 (CH, C3), 127.3(CH), 128.8 (CH), 130.3 (CH), 132.6 (CH), 175.6 (CO, C24) ppm.

IR=3253 (OH), 2929 (alkyl), 2865 (alkyl), 1637 (C═O), 1565, 1441 cm⁻¹.

MS (+ESI) m/z=Found 551.4570 M⁺; calculated for C₃₆H₅₉N₂O₂ 551.4571; 0.2ppm.

Synthesis of3-[[(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]propyl-dimethyl-[(4-vinylphenyl)methyl]ammoniumchloride (134)

Compound 103 (1.0 g 0.001 mol) was dissolved in dichloromethane (20 mL).Vinyl benzyl chloride (0.89 mL, 0.005) was added and the solution wasstirred for 48 hours at ambient temperature. The resulting precipitatewas collected by vacuum filtration and was washed with petroleum ether60/80 (3×20 mL). The resultant crude product was dissolved in methanoland again washed with petroleum ether 60/80 (3×20 mL).

The solvent was removed under reduced pressure to produce off-whitesolid.

Yield; 1 g, 0.001 mol, 75%.

Melting point: >350° C.

¹H NMR (MeOD) (250 MHz) δ=0.67 (s, 3H, 18-CH₃), 0.94 (s, 3H, 19-CH₃),3.05 (s, 6H, 2×CH₃), 4.54 (s, 2H, CH₂), 5.37 (d, 1H, ═CH—, J=12.5), 5.91(d, 2H, ═CH, J=15.0), 6.80 (dd, 1H, ═CH, J=12.5), 7.56 (q, 5H, Ar—CH,J=7.5) ppm.

¹³C NMR (62.9 MHz) δ=11.9 (CH₃, C18), 18.2 (CH₃, C21), 20.6 (CH₂, C11),23.3 (CH₃, C19), 24.0 (CH₂, C15), 26.3 (CH₂, C7), 27.1 (CH₂, C6), 28.0(CH₂, C16), 30.3 (CH₂, C2), 30.9 (CH₂, C22), 30.9 (CH₂, C23), 34.2 (C,C10), 35.1 (CH, C20), 35.3 (CH₂, C1), 35.6 (CH, C8), 36.3 (CH₂, C4),40.0 (CH₂, C12), 40.2 (CH, C9), 41.9 (CH, C5), 42.4 (C, C13), 55.8 (CH,C17), 56.3 (CH, C14), 70.5 (CH, C3), 178.1 (CO, C24) ppm.

IR=3356 (OH), 2929 (alkyl), 2852 (alkyl), 1633 (C═O), 1548, 1441, 1031(R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 577.4723 M⁺; calculated for C₃₈H₆₁N₂O₂ 577.4728; 0.8ppm.

Synthesis of(4R)-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-N-[2-(1-methylpyrrolidin-1-ium-1-yl)ethyl]pentanamideiodide (135)

Compound 108 (0.1 g 0.0002 mol) was dissolved in chloroform (10 mL).Methyl iodide (0.09 mL 0.0006 mol) was added and the solution wasstirred for 1 week. The solvent was evaporated under reduced pressure.The residue was re-dissolved in methanol and washed with petroleum ether60/80 (3×20 mL). The methanol phase was evaporated under reducedpressure to give the product as a green-brown oil was produced.

Yield; 0.08 g, 0.0001 mol, 66%.

¹H NMR (CDCl₃) (250 MHz) δ=0.70 (s, 3H, 18-CH₃), 0.93 (s, 3H, 19-CH₃),1.00 (d, 3H, 21-CH₃ J=10.0), 3.15 (s, 3H, CH₃), 3.50-3.70 (multipleoverlapping multiplets, 7H, CH/CH₂), 3.95 (s, 1H, 12-CH) ppm.

¹³C NMR (250 MHz) δ=13.2 (CH₃, C18), 17.7 (CH₃, C21), 22.56 (CH₂) 23.7(CH₃, C19), 24.9 (CH₂, C15), 27.5 (CH₂, C7), 28.44 (CH₂, C6), 28.7 (CH₂,C16), 29.9 (CH₂, C11), 31.1 (CH₂, C2), 33.0 (CH₂, C23), 33.9 (CH₂, C22),34.8 (CH, C9), 35.3 (C, C10), 36.4 (CH₂, C1), 36.9 (CH, C20), 37.2 (CH₂,C4), 37.4 (CH, C8), 43.6 (CH, C5), 63.5 (CH₂), 66.1 (CH₂), 72.5 (CH,C3), 74.0 (CH₂, C12), 176.5 (CO, C24) ppm.

IR=3394 (OH), 2916 (alkyl), 2857 (alkyl), 1646 (C═O), 1530, 1445, 1368,1253, 1036 (RCH—OH) cm⁻¹.

MS (+ESI) m/z=Found 503.4201 M⁺; calculated for C₃₁H₅₅N₂O₃ 503.4207; 1.2ppm.

Synthesis of(4R)—N-[2-(1-allylpyrrolidin-1-ium-1-yl)ethyl]-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanamidebromide (136)

Compound 107 (0.5 g 0.001 mol) was dissolved in dichloromethane (15 mL).Ally bromide (2.79 mL, 0.02) was added and the solution was stirred for48 hours at 50° C. The resulting precipitate was collected by vacuumfiltration, triturated chloroform (3×20 mL) and dried under vacuum.

Yield; 0.168 g, 0.0002 mol, 27%.

Melting point: 211.6-214.8° C.

¹H NMR (MeOD) (250 MHz) δ=0.68 (s, 3H, 18-CH₃), 0.94 (s, 3H, 19-CH₃),3.62 (overlapping mutliplets, 4H, CH₂), 4.02 (d, 2H, CH₂, J=7.5), 5.74(multiple overlapping multiplets, 2H, ═CH₂), 6.11 (m, 1H, ═CH—) ppm.

¹³C NMR (62.9 MHz) δ=11.0 (CH₃, C18), 17.3 (CH₃, C21), 20.4 (CH₂, C11),22.3 (CH₃, C19), 22.4 (CH) 23.7 (CH₂, C15), 26.1 (CH₂, C7), 26.8 (CH₂,C6), 27.7 (CH₂, C16), 29.6 (CH₂, C2), 31.4 (CH₂, C22), 32.3 (CH₂, C23),33.2 (C, C10), 34.1 (CH, C20), 34.9 (CH₂, C1), 35.3 (CH, C8), 35.6 (CH₂,C4), 40.0 (CH₂, C12), 40.3 (CH, C9), 42.0 (CH, C5), 42.4 (C, C13), 55.6(CH, C17), 56.4 (CH, C14), 57.8 (CH₂), 61.3 (CH₂), 62.1 (CH₂) 70.8 (CH,C3), 125.2 (CH), 127.3 (CH₂) 175.8 (CO, C24) ppm.

IR=3411 (NH), 3198 (OH), 2925 (alkyl), 2852 (alkyl), 1637 (C═O), 1560,1441, 1368, 1066 (R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 513.4405 M⁺; calculated for C₃₃H₅₇N₂O₂ 513.4415; 1.9ppm.

Synthesis of(4R)—N-[2-(1-allylpyrrolidin-1-ium-1-yl)ethyl]-4-[(3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanamidebromide (137)

Compound 108 (0.1 g 0.0002 mol) was dissolved in chloroform (10 mL) withallyl bromide (0.08 mL, 0.0006 mol). The solution was stirred for 1week. The solvent was evaporated under reduced pressure and the productwas re-dissolved in methanol and washed with petroleum ether (3×10 mL).The solvent was then removed under reduced pressure to produce a whitesolid.

Yield; 0.1 g, 0.0001 mol, 83%.

Melting point: 176.2-179.8° C.

¹H NMR (MeOD) (250 MHz) δ=0.70 (s, 3H, 18-CH₃), 0.93 (s, 3H, 19-CH₃),1.02 (d, 3H, 21-CH₃, J=5.0), 2.22 (broad s, 4H, CH₂), 3.515 (q, 2H,CH₂), 3.65 (m, 3H, CH₂/3-CH), 4.01 (broad s, 1H, 12-CH), 4.025 (d, 2H,CH₂ J=7.5), 5.74 (m, 2H, CH₂), 6.11 (m, 1H, ═CH—) ppm.

¹³C NMR (62.9 MHz) δ=12.4 (CH₃, C18), 16.8 (CH₃, C21), 21.8 (CH₂), 22.9(CH₃, C19), 24.0 (CH₂, C15), 26.6 (CH₂), 27.6 (CH₂, C7), 27.9 (CH₂, C6),28.1 (CH₂, C16), 30.9 (CH₂, C2), 32.2 (CH₂, C23), 33.0 (CH₂, C22), 34.0(CH, C9), 36.0 (CH₂), 36.6 (CH, C20), 42.7 (CH, C5), 44.8 (C, C13), 47.1(CH, C17), 58.4 (CH₂), 61.9 (CH₂), 62.8 (CH₂) 71.7 (CH, C3), 73.1 (CH₂,C12), 126.7 (CH₂), 128.9 (CH₂) 177.4 (CO, C24) ppm.

IR=3305 (OH), 2921 (alkyl), 2857 (alkyl), 1641 (C═O), 1539, 1449, 1040(R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 529.4349 M⁺; calculated for C₃₃H₅₇N₂O₃, 529.4364;2.8 ppm.

Synthesis of(4R)—N-[2-(1-benzylpyrrolidin-1-ium-1-yl)ethyl]-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanamidebromide (138)

Compound 107 (0.5 g 0.001 mol) was dissolved in dichloromethane (20 mL).Benzyl bromide (1.42 mL 0.008 mol) was added and the solution wasstirred for 24 hours. The resultant precipitate was collected by vacuumfiltration to produce a white powder.

Yield; 0.01 g, 0.0001 mol, 1.5%.

Melting point: 216.2-220.8° C.

¹H NMR (CDCl₃) (250 MHz) δ=0.66 (s, 3H, 18-CH₃), 0.94 (s, 3H, 19-CH₃),3.54-3.76 (multiple overlapping multiplets, 7H, 3-CH/CH₂), 4.57 (s, 2H,CH₂), 7.56 (m, 5H, Ar—CH) ppm.

¹³C NMR (62.9 MHz) δ=10.9 (CH₃, C18), 17.3 (CH₃, C21), 20.4 (CH₂), 20.7(CH₂, C11), 22.4 (CH₃, C19), 23.7 (CH₂, C15), 27.7 (CH₂, C16), 29.6(CH₂, C2), 31.5 (CH₂, C22), 32.3 (CH₂, C23), 34.1 (C, C10), 34.9 (CH,C20), 35.3 (CH₂, C1), 35.6 (CH, C8), 35.7 (CH₂, C4), 40.0 (CH₂, C12),40.3 (CH, C9), 41.9 (CH, C5), 42.4 (C, C13), 55.8 (CH, C17), 56.4 (CH,C14), 57.0 (CH), 61.2 (CH), 70.8 (CH, C3), 127.7 (CH), 129.0 (CH), 130.4(CH), 132.2 (CH) 175.8 (CO, C24) ppm.

IR=3343 (NH), 3241 (OH), 2929 (alkyl), 2844 (alkyl), 1667 (C═O), 1535,1445, 1360, 1070 (R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 563.4563 M⁺; calculated for C₃₇H₅₉N₂O₂ 563.4571; 1.4ppm.

Synthesis of(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-N-[2-[1-[(4-vinylphenyl)methyl]pyrrolidin-1-ium-1-yl]ethyl]pentanamidechloride (139)

Compound 107 (0.5 g 0.001 mol) was dissolved in dichloromethane (20 mL).Vinyl benzyl chloride (1.74 mL 0.01 mol) was added and the solution wasstirred for 48 hours at ambient temperature followed by heating at 50°C. for 15 h. The solvent was evaporated under reduced pressure. Thematerial re-dissolved in methanol and washed with petroleum ether 60-80(3×20 mL). A white solid was produced. This was collected by vacuumfiltration and dried under vacuum.

Yield: 0.08 g, 0.0001 mol, 12%.

Melting point: >350° C.

¹H NMR (CDCl₃) (250 MHz) δ=0.65 (s, 3H, 18-CH₃), 0.94 (s, 3H, 19-CH₃),3.56 (multiple overlapping multiplets, 4H, CH₂), 4.57 (s, 2H, CH₂), 5.37(d, 1H, ═CH—, J=12.5), 5.90 (d, 1H, ═CH, J=17.5), 6.80 (dd, 1H, ═CH,J=12.5), 7.60 (s, 4H, Ar—CH) ppm.

¹³C NMR (62.9 MHz) δ=10.9 (CH₃, C18), 17.3 (CH₃, C21), 20.4 (CH₂, C11),20.7 (CH₂), 22.4 (CH₃, C19), 23.7 (CH₂, C15), 26.1 (CH₂, C7), 27.7 (CH₂,C16), 29.6 (CH₂, C2), 31.5 (CH₂, C22), 32.3 (CH₂, C23), 33.2 (C, C10),34.1 (CH, C20), 34.9 (CH₂, C1), 35.3 (CH, C8), 35.7 (CH₂, C4), 40.0(CH₂, C12), 40.3 (CH, C9), 41.9 (CH, C5), 42.4 (C, C13), 55.8 (CH, C17),56.4 (CH, C14), 57.0 (CH₂), 61.2 (CH), 61.8 (CH₂) 70.8 (CH, C3), 115.0(CH), 126.6 (CH), 132.5 (CH), 135.4 (CH), 139.9 (CH), 175.8 (CO, C24)ppm.

IR=3360 (OH), 2925 (alkyl), 2852 (alkyl), 1641 (C═O), 1539, 1437, 1368,1036 (R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 589.4719 M⁺; calculated for C₃₉H₆₁N₂O₂ 589.4728; 1.5ppm.

Synthesis of(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-1-[4-(2-hydroxyethyl)-4-methyl-piperazin-4-ium-1-yl]pentan-1-oneiodide (140)

Compound 118 (0.2 g 0.0004 mol) was dissolved in chloroform (5 mL).Iodomethane (0.29 mL, 0.002 mol) was added and the solution was stirredovernight at ambient temperature. The resulting precipitate wascollected by vacuum filtration and was dried overnight in a desiccator.The product was an off-white solid.

Yield; 0.01 g, 0.00001 mol, 4%.

Melting point: 245.3-251.3° C.

¹H NMR (MeOD) (250 MHz) δ=0.69 (s, 3H, 18-CH₃), 0.94 (s, 3H, 19-CH₃),0.98 (d, 3H, 21-CH₃, J=5.0), 3.54-3.65 (multiple overlapping multiplets,6H, CH₂), 4.01 (multiple overlapping multiplets, 6H, CH₂) ppm.

¹³C NMR (62.9 MHz) δ=11.5 (CH₃, C18), 16.4 (CH₃, C21), 22.9 (CH₂, C11),25.9 (CH₂), 27.8 (CH₂), 32.2 (CH), 33.2 (CH), 34.6 (C, C10), 35.1 (CH,C20), 41.1 (CH, C5), 42.4 (C, C13), 44.5 (CH₂), 45.8 (CH), 47.4 (CH),60.4 (CH₂), 61.3 (CH), 61.8 (CH), 67.1 (CH₂), 70.1 (CH, C3), 71.2 (CH₂),75.7 (CH₂), 76.3 (CH₂), 80.6 (CH₂) 174.8 (CO, C24) ppm.

IR=3313 (OH), 2929 (alkyl), 2852 (alkyl), 1607 (C═O), 1466, 1249, 1044(R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 503.4195 M⁺; calculated for C₃₁H₅₅N₂O₃ 503.4207; 2.4ppm.

Synthesis of(4R)-1-[4-allyl-4-(2-hydroxyethyl)piperazin-4-ium-1-yl]-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentan-1-onebromide (141)

Compound 118 (0.2 g 0.0004 mol) was dissolved in chloroform (5 mL).Allyl bromide (0.44 mL, 0.003 mol) was added and the solution wasstirred overnight at ambient temperature. The resulting precipitate wascollected by vacuum filtration and was dried overnight in a desiccator.The product was an off-white solid.

Yield; 0.02 g, 0.00003 mol, 8%.

Melting point: 194.9-197.8° C.

¹H NMR (MeOD) (250 MHz) δ=0.70 (s, 3H, 18-CH₃), 0.94 (s, 3H, 19-CH₃),0.98 (d, 3H, 21-CH₃, J=7.5), 3.54-3.65 (multiple overlapping multiplets,6H, CH₂), 4.01 (multiple overlapping multiplets, 6H, CH₂), 4.27 (d, 2H,CH₂, J=7.5), 5.76 (dd, 2H, ═CH₂), 6.10 (m, 1H, ═CH—) ppm.

¹³C NMR (62.9 MHz) δ=12.5 (CH₃, C18), 18.9 (CH₃, C21), 21.9 (CH₂, C11),23.9 (CH₃, C19), 25.3 (CH₂, C15), 27.6 (CH₂, C6), 28.3 (CH₂, C16), 29.3(CH₂, C2), 30.6 (CH₂, C22), 31.1 (CH₂, C23), 32.2 (C, C10), 35.6 (CH,C20), 36.4 (CH₂, C1), 36.9 (CH, C8), 37.2 (CH₂, C4), 41.5 (CH₂, C12),41.9 (CH, C9), 56.4 (CH, C17), 57.4 (CH, C14), 57.9 (CH₂), 59.4 (CH₂),61.6 (CH₂), 63.7 (CH₂) 72.4 (CH, C3), 125.6 (CH), 129.9 (CH), 174.8 (CO,C24) ppm.

IR=3350 (NH), 3241 (OH), 2938 (alkyl), 2850 (alkyl), 1633 (C═O), 1439,1244, 1189.78 (C═O ester stretch), 1244 (R₂CH—OH) cm⁻¹.

MS (ES) m/z=Found 529.4349 M⁺; calculated for C₃₃H₅₇N₂O₃ 529.4364; 2.8ppm.

Synthesis of(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-1-[4-(2-hydroxyethyl)-4-pentyl-piperazin-4-ium-1-yl]pentan-1-onebromide (142)

Compound 103 (0.5 g 0.001 mol) was dissolved in dichloromethane (20 mL).Iodopentane (1.57 mL, 0.007 mol) was added and the solution was stirredovernight at 40° C. The resulting precipitate was collected by vacuumfiltration and was dried overnight in a desiccator. The product was anoff-white solid.

Yield; 0.36 g, 0.0005 mol, 50.7%.

Melting point: 104.8-108.6° C.

¹H NMR (MeOD) (250 MHz) δ=0.66 (s, 3H, 18-CH₃), 0.92 (s, 3H, 19-CH₃),0.94 (multiple overlapping multiplets consisting of both CH₃ and CH₂),3.08 (s, 5H, CH₃/CH₂). 3.51 (m, 1H, 3-CH) ppm.

¹³C NMR (62.9 MHz) δ=11.0 (CH₃, C18), 12.7, 17.3 (CH₃, C21), 20.4 (CH₂,C11), 21.8 (CH₃, C19), 26.1 (CH₂, C7), 29.6 (CH₂, C2), 34.1 (C, C10),35.4 (CH, C20), 35.7 (CH₂, C1), 40.0 (CH₂, C12), 40.4 (CH, C9), 42.0(CH, C5), 42.4 (C, C13), 49.8, 55.9 (CH, C17), 56.4 (CH, C14), 70.8 (CH,C3), 175.7 (CO, C24) ppm.

IR=3449 (OH), 2921 (alkyl), 2857 (alkyl), 1641 (C═O), 1548, 1445, 1364,1036 (R₂CH—OH) cm⁻¹.

MS (+ESI) m/z=Found 531.4880 M⁺; calculated for C₃₄H₆₃N₂O₂ 531.4884; 0.8ppm.

Synthesis of(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-1-[4-(2-hydroxyethyl)-4-[(4-vinylphenyl)methyl]piperazin-4-ium-1-yl]pentan-1-onechloride (143)

Compound 118 (0.2 g 0.0004 mol) was dissolved in chloroform (5 mL).Vinyl benzyl chloride (0.31 mL, 0.002 mol) was added and the solutionwas stirred overnight at ambient temperature. The resulting precipitatewas collected by vacuum filtration and was dried overnight in adesiccator. The product was an off-white solid.

Yield; 0.1 g, 0.0001 mol, 38%.

Melting point: 143.6-146.0° C.

¹H NMR (MeOD) (250 MHz) δ=0.680 (s, 3H, 18-CH₃), 0.94 (s, 3H, 19-CH₃),3.61 (multiple overlapping multiplets, 6H, CH₂), 5.37 (d, 1H, ═CH—,J=12.5), 5.90 (d, 1H, ═CH, J=17.5), 6.80 (dd, 1H, ═CH, J=12.5), 7.59(broad s, 4H, Ar—CH) ppm.

¹³C NMR (62.9 MHz) δ=12.5 (CH₃, C18), 18.9 (CH₃, C21), 21.9 (CH₂, C11),23.9 (CH₃, C19), 25.3 (CH₂, C15), 27.6 (CH₂, C7), 28.3 (CH₂, C6), 29.3(CH₂, C16), 30.6 (CH₂, C2), 31.2 (CH₂, C22), 32.2 (CH₂, C23), 35.6 (C,C10), 36.4 (CH, C20), 36.8 (CH₂, C1), 37.2 (CH, C8), 40.6 (CH₂, C12),41.5 (CH, C9), 41.9 (CH, C5), 43.5 (C, C13), 56.5 (CH, C17), 57.4 (CH,C14), 57.9 (CH), 58.1 (CH₂), 58.8 (CH₂) 67.1 (CH) 72.4 (CH, C3), 116.7(CH), 127.2 (CH), 128.0 (CH), 134.9 (CH), 137.0 (CH), 141.6 (CH), 174.8(CO, C24) ppm.

IR=3283 (OH), 2925 (alkyl), 2857 (alkyl), 1616 (C═O), 1445, 1044(R₂CH—OH) cm⁻¹.

MS (ES) m/z=Found 605.4669 M⁺; calculated for C₃₉H₆₁N₂O₃ 605.4677; 1.3ppm.

Germination Tests

Many of the synthesised compounds were tested for their germinating andantimicrobial abilities against C. difficile. Due to the insolubility ofsome of the compounds in water, DMSO, ethanol and methanol were used todissolve the compounds.

Methods

The C. difficile reference strain, NCTC 11204 and C. difficile ribotype027 (R20291) (Anaerobic Reference Laboratory, Cardiff, UK) were usedduring testing.

Preparation of Spore Suspensions

Spore suspensions of C. difficile were prepared following the methodproposed by Shetty et al. (1999). Briefly, Columbia base agar plateswere inoculated with the relevant strain of C. difficile and incubatedfor 72 hours anaerobically at 37° C. (MiniMACS anaerobic cabinet, DonWhitley Scientific, Shipley, UK). Then, the plates were removed and leftor 24 hours in aerobic conditions at room temperature. Colonies werethen harvested into 20 mL of 50% (w/v) ethanol and 50% saline, andvortex thoroughly. These were stored at 4° C. until needed.

All experiments were performed in triplicate using spore suspensionscontaining 1×10⁷ CFU mL⁻¹ spores of C. diffcile NCTC 11204 and ribotype027.

Before use, 1 mL of spores were centrifuged at 13000 rpm for 10 minutes(Spectrafuge 24D; Labnet, Woodbridge, USA). The supernatant wasdiscarded, and the pellet resuspended in 1 mL sterile distilled waterand vortex mixed thoroughly.

Germination solutions were prepared using 2% (w/v) of the compound indiluent (DMSO, ethanol, methanol, water) plus double strengththioglycollate medium (Oxoid, UK).

Heat Shock Method

For the heat shock method, 100 μl spores were exposed to 100 μl of thegermination solution and incubated at room temperature in air for 1hour. The entire 200 μl sample was then added to 800 μl steriledistilled water to dilute out the germinant to ineffectiveconcentrations. Samples were placed on heat at 70° C. for 20 minutes toeliminate any germinated, metabolically active spores. Control sampleswere kept on ice. Solutions were then diluted accordingly using steriledistilled water, and cultured onto fastidious anaerobic agar (Lab M,Bury, UK), supplemented with 0.1% (w/v) sodium taurocholate (ST) and 5%(w/v) defibrinated horse blood using the Miles and Misra method (Mileset al., 1938). These were then incubated anaerobically for 48 hours at37° C. (MiniMACS anaerobic cabinet, Don Whitley Scientific, Shipley, UK)and the CFU mL⁻¹ counted.

Germination results for strains 11204 and 027 >1 log >1 log CompoundCompound reduction reduction number name Structure on heat on ice 4 3-cholanamidopropyl- allyl-dimethyl- ammonium bromide

yes X 50 N-[2-(1- benzylpyrrolidin-1- ium-1-yl)ethyl] cholanamidebromide

yes yes 29 3-cholan amidopropyl- hexyl-dimethyl- ammonium iodide

yes X 45 N-[2-(1- propylpyrrolidin- 1-ium-1- yl)ethyl] cholanamideiodide

yes yes 16 N-octadecyl cholanamide

Yes (11204 and 027) X 7 N-[3- (dibutylamino) propyl] cholanamide

Yes (11204) Yes (11204) 2 cholic acid benzyl amide

X Yes (11204) 2/150

X Yes (11204) 2/148

Yes (027) x 10 N-(2-pyrrolidin- 1-ylethyl) cholanamide

Yes (11204) X

The results from the table above show the compounds that gave a 1 logreduction or more between the initial spore count and the spore countafter the heat and ice treatment. A reduction in the heat count suggeststhe compound is a germinant, whereas a reduction in the ice suggests asporicide or a germinant/antimicrobial compound. Due to the nature ofthis test, it is difficult to distinguish between the two.

The invention claimed is:
 1. A method of treating a surface containingC. difficile spores, comprising exposing the surface to a compoundselected from the group consisting of: 2-[[(4R)-4-[(3R,10S, 13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]ethyl-dimethyl-[(4-vinylphenyl)methyl]ammoniumchloride; 3-cholanamidopropyl-allyl-dimethyl-ammonium bromide;N-[2(1-benzylpyrrolidin-1-ium-1-yl)ethyl] cholanamide bromide;3-cholanamidopropyl-hexyl-dimethylammonium iodide; andN-[2-(1-propylpyrrolidin-1-ium-1-yl)ethyl] cholanamide iodide.
 2. Themethod according to claim 1 for use in the prevention of a diseaseselected from: C. difficile associated disease (CDAD) and toxicmegacolon.
 3. A method of treating a surface containing C. difficilespores comprising exposing the surface to a composition comprising: acompound selected from the group consisting of: 2-[[(4R)-4-[(3R,10S,13R,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]ethyl-dimethyl-[(4-vinylphenyl)methyl]ammoniumchloride; 3-cholanamidopropyl-allyl-dimethyl-ammonium bromide;N-[2-(1-benzylpyrrolidin-1-ium-1-yl)ethyl] cholanamide bromide;3-cholanamidopropyl-hexyl-dimethylammonium iodide; and N-[2-(1-propylpyrrolidin-1-ium-1-yl)ethyl] cholanamide iodide.